Modulation of structured polypeptide specificity

ABSTRACT

The invention describes peptide ligands specific for human plasma Kallikrein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase filing under 35 U.S.C. 371 ofInternational Patent Application No. PCT/EP/2014/057440, filed Apr. 11,2014, which claims priority to British application number GB 1306623.8,filed Apr. 11, 2013.

The present invention relates to polypeptides which are covalently boundto molecular scaffolds such that two or more peptide loops are subtendedbetween attachment points to the scaffold. In particular, the inventiondescribes peptides which are specific for the human protease plasmaKallikrein and are modified in one or two peptide loops to enhancepotency and/or protease resistance.

Cyclic peptides are able to bind with high affinity and targetspecificity to protein targets and hence are an attractive moleculeclass for the development of therapeutics. In fact, several cyclicpeptides are already successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug octreotide (Driggers, et al., NatRev Drug Discov 2008, 7 (7), 608-24). Good binding properties resultfrom a relatively large interaction surface formed between the peptideand the target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu, B., et al., Science 330 (6007), 1066-71),a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3(355 Å²) (Xiong, J. P., et al., Science 2002, 296 (5565), 151-5) or thecyclic peptide inhibitor upain-1 binding to urokinase-type plasminogenactivator (603 Å²; Zhao, G., et al., J Struct Biol 2007, 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8, MMP-8) which lost its selectivity over other MMPswhen its ring was opened (Cherney, R. J., et al., J Med Chem 1998, 41(11), 1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin oractinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp, D. S. andMcNamara, P. E., J. Org. Chem, 1985; Timmerman, P. et al., ChemBioChem,2005). Meloen and co-workers had used tris(bromomethyl)benzene andrelated molecules for rapid and quantitative cyclisation of multiplepeptide loops onto synthetic scaffolds for structural mimicry of proteinsurfaces (Timmerman, P. et al., ChemBioChem, 2005). Methods for thegeneration of candidate drug compounds wherein said compounds aregenerated by linking cysteine containing polypeptides to a molecularscaffold as for example tris(bromomethyl)benzene are disclosed in WO2004/077062 and WO 2006/078161.

WO2004/077062 discloses a method of selecting a candidate drug compound.In particular, this document discloses various scaffold moleculescomprising first and second reactive groups, and contacting saidscaffold with a further molecule to form at least two linkages betweenthe scaffold and the further molecule in a coupling reaction.

WO2006/078161 discloses binding compounds, immunogenic compounds andpeptidomimetics. This document discloses the artificial synthesis ofvarious collections of peptides taken from existing proteins. Thesepeptides are then combined with a constant synthetic peptide having someamino acid changes introduced in order to produce combinatoriallibraries. By introducing this diversity via the chemical linkage toseparate peptides featuring various amino acid changes, an increasedopportunity to find the desired binding activity is provided. FIG. 1 ofthis document shows a schematic representation of the synthesis ofvarious loop peptide constructs. The constructs disclosed in thisdocument rely on —SH functionalised peptides, typically comprisingcysteine residues, and heteroaromatic groups on the scaffold, typicallycomprising benzylic halogen substituents such as bis- ortris-bromophenylbenzene. Such groups react to form a thioether linkagebetween the peptide and the scaffold.

We recently developed a phage display-based combinatorial approach togenerate and screen large libraries of bicyclic peptides to targets ofinterest (Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7; see alsointernational patent application WO2009/098450). Briefly, combinatoriallibraries of linear peptides containing three cysteine residues and tworegions of six random amino acids (Cys-(Xaa)₆-Cys-(Xaa)_(e)-Cys) (SEQ IDNo. 210) were displayed on phage and cyclised by covalently linking thecysteine side chains to a small molecule (tris-(bromomethyl)benzene).Bicyclic peptides isolated in affinity selections to the human proteasescathepsin G and plasma Kallikrein (PK) had nanomolar inhibitoryconstants. The best inhibitor, PK15, inhibits human PK (hPK) with aK_(l) of 3 nM. Similarities in the amino acid sequences of severalisolated bicyclic peptides suggested that both peptide loops contributeto the binding. PK15 did not inhibit rat PK (81% sequence identity) northe homologous human serine proteases factor XIa (hfXIa; 69% sequenceidentity) or thrombin (36% sequence identity) at the highestconcentration tested (10 μM) (Heinis, et al., Nat Chem Biol 2009, 5 (7),502-7). This finding suggested that the bicyclic inhibitor is highlyspecific and that other human trypsin-like serine proteases will not beinhibited. A synthetic, small peptidic inhibitor such as PK15 having theabove described potency and target selectivity has potential applicationas a therapeutic to control PK activity in hereditary angioedema, alife-threatening disease which is characterized by recurrent episodes ofedema or to prevent contact activation in cardiopulmonary bypasssurgery.

The peptide PK15 was isolated from a library based on the peptide PK2,H-ACSDRFRNCPLWSGTCG-NH₂ (SEQ ID No. 1), in which the second 6-amino acidloop was randomised. The sequence of PK15 was H-ACSDRFRNCPADEALCG-NH₂(SEQ ID No. 2), and the IC50 binding constant for human Kallikrein was1.7 nM.

SUMMARY OF THE INVENTION

We have analysed kallikrein-binding reagents in order to optimisebinding affinity and activity. In our copending unpublished patentapplication PCT/EP2012/069898, selective bicyclic peptide ligands whichare Inhibitors of human plasma kallikrein have been described.

In one embodiment described therein, the loops of the peptide ligandcomprise five amino acids and a first loop comprises the motifG_(r)x^(W)/_(F)Px^(K)/_(R)G_(r) (SEQ ID No. 211), wherein G_(r) is areactive group. In the present context, the reference to a “first” loopdoes not necessarily denote a particular position of the loop in asequence. In some embodiments, however, the first loop may be proximalloop in an amino terminus to carboxy terminus peptide sequence. Forexample, the polypeptide further comprises a second, distal loop whichcomprises the motif G_(r) ^(T)/_(L)H^(Q)/_(T)xLG_(r) (SEQ ID No. 212).Examples of sequences of the first loop include G_(r)XWPARG_(r) (SEQ IDNo. 3), G_(r)xWPSRG_(r) (SEQ ID No. 4), G_(r)xFPFRG_(r) (SEQ ID No. 5)and G_(r)xFPYRG_(r) (SEQ ID No. 6). In these examples, x may be anyamino acid, but is for example S or R.

For example, the polypeptide may be one of the polypeptides set forth inTable 4, Table 5 or Table 6.

The reactive group can be a reactive amino acid. For example, thereactive amino acid is cysteine.

Variants of the polypeptides can be prepared by identifying thoseresidues which are available for mutation and preparing libraries whichinclude mutations at those positions. For example, the polypeptide 06-56in Table 4, FIGS. 5, 6 can be mutated without loss of activity atpositions Q4 and T10 (see Examples below). Polypeptide ligandscomprising mutations at these positions can be selected which haveimproved binding activity in comparison with 06-56.

For a kallikrein-inhibiting bicycle, it is pertinent to obtain anadequate protease stability profile, such that it has a lowprotease-driven clearance in plasma or other relevant environments. In arapid comparative plasma stability assay (Method #1) that observed theprogressive disappearance of parent peptide in rat plasma, it was foundthat the N-terminal alanine (which is present at the time of selectionsand was originally included in synthetic peptides of lead sequences) israpidly removed across all bicycle sequences tested by both rat andhuman plasma. This degradation was avoided by synthesising a leadcandidate lacking both N- and C-terminal alanines. To remove potentialrecognition points for amino- and carboxypeptidases, the freeamino-terminus that now resides on Cys 1 of the lead candidate is cappedwith acetic anhydride during peptide synthesis, leading to a moleculethat is N-terminally acetylated. In an equal measure, the C-terminalcysteine is synthesised as the amide so as to remove a potentialrecognition point for carboxypeptidases.

Thus, in one example Bicyclic lead candidates have the following genericsequence:

Ac-C ₁ AA ₁ AA ₂ AA _(n) C ₂ AA _(n+1) AA _(n+2) AA _(n+3) C ₃(TMB)-NH₂where “Ac” refers to N-terminal acetylation, “—NH₂” refers to C-terminalamidation, where “C₁, C₂, C₃” refers to the first, second and thirdcysteine in the sequence, where “AA₁” to “AA_(n)” refers to the positionof the amino acid (whose nature “AA” is defined by the selectionsdescribed above), and where “(TMB)” indicates that the peptide sequencehas been cyclised with TBMB (trisbromomethylbenzene) or any othersuitable reactive scaffold.

In this context, the lead peptides “Ac-(06-34-18)(TMB)” and“Ac-(06-34-18)(TMB) Phe2 Tyr4” have the sequences Ac-CSWPARCLHQDLC-NH₂(SEQ ID No. 7) and Ac-CSFPYRCLHQDLC-NH₂ (SEQ ID No. 8), respectively.These can be numbered according to the above scheme as

(SEQ ID No. 7) 1) Ac-C ₁S₁W₂P₃A₄R₅ C ₂L₆H₇Q₈D₉L₁₀ C ₃-NH₂denoted as Ac-(06-34-18)(TMB) (SEQ ID No. 8) 2) Ac-C ₁S₁F₂P₃Y₄R₅ C₂L₆H₇Q₈D₉L₁₀ C ₃-NH₂ denoted as Ac-(06-34-18)(TMB) Phe2 Tyr4where the invariant cysteines are underlined. These peptides have beencharacterised in detail, and shown to have sub-nanomolar potency againstkallikrein, with very favourable selectivity profiles towardskallikrein-homologous proteins, while retaining good cross-reactivitytowards rat kallikrein. The latter is valuable for establishingpharmacodynamics in animal models.

The unmodified peptide Ac-06-34-18(TMB)-NH₂ has a half-life of 2.3 hrsin rat plasma, while that of Ac-06-34-18(TMB)-NH₂ Phe2 Tyr4 is slightlyshorter, at 0.9 hrs (Table 1). In an effort to identify the proteolyticrecognition site(s) in Ac-06-34-18(TMB)-NH₂, the peptide was sampled inrat plasma over time (method #1), and each sample was analysed for theprogressive appearance of peptide fragments using MALDI-TOF massspectrometry. This revealed Arg5 in Loop 1 to be the main site ofprotease recognition and subsequent peptide degradation.

A process of chemical synthesis was undertaken to identify Arginine5substitutes that would sufficiently protect the peptide from rat plasmaprotease degradation. It was found that removal of Arg 5 in loop 1, orreplacement of the Arg side chain with any non-charged or chargedchemical entities, enhances the stability of the peptide ligand.

Moreover, the proteolytic stability is enhanced by chemical modificationof the peptide bonds N- or C-terminally adjacent to Arg5. The chemicalmodification is, for example, α-N-alkylation, or modification of thepeptide bond with its reduced amide form.

In one embodiment, proteolytic stability is enhanced by stericobstruction of nearby amino acids to Arg5. For example, the stericobstruction results from α-N-alkylation.

These Bicyclic Peptide candidates were then evaluated for the followingcharacteristics:

-   -   1) maintenance of potency against human kallikrein (seeking a Ki        of 10 nM or lower)    -   2) maintenance of potency against rat kallikrein (seeking a Ki        of 50 nM or lower)    -   3) enhanced stability in rat plasma (greater t_(1/2) desirable        compared to the wildtype peptide)    -   4) maintenance of selectivity profile against other        kallikrein-related enzymes and proteins.

In the course of the investigations in PCT/EP2012/069898, it wasdetermined that the following Arginine modifications or mimics fulfilledthe 4 above criteria, these being

a) 4-guanidylphenylalanine (4-GuanPhe: t_(1/2) increase in rat plasma:1-5 fold);

b) homoarginine (HArg: t_(1/2) increase in rat plasma: ˜2-5-fold); and

c) α-N-methylarginine (NMe-Arg: t_(1/2) increase in rat plasma:>10-fold)

We have found that certain non-natural amino acids permit binding toplasma Kallikrein with nM Ki, whilst increasing residence time in plasmasignificantly. The data are summarized in Table 1 and FIG. 17.

Moreover, PCT/EP2012/069898 also disclosed an additional modification atposition 3 in 06-34-18, where the proline was replaced with azetidinecarboxylic acid (Aze3). This modification, likely due to its greaterconstraint and reduced entropy, invariably enhances the potency of an06-34-18-based peptide by a factor of 2 to 5 (Table 1). In combinationwith the stability enhancing modifications at Arg 5 (specifically, HArg,NMe-Arg and 4GuanPhe), more potent Bicyclic Peptide derivatives areobtained.

Exemplary non-natural amino acids are selected from N-methyl Arginine,homo-arginine and hydroxyproline. N-methyl and homo-derivatives ofArginine are used to replace Arginine, and proline 3 can be replaced by,for example, hydroxyproline, azetidine carboxylic acid, or analpha-substituted amino acid, such as aminoisobutyric acid. In anotherembodiment, arginine may be replaced with guanidyl-phenylalanine.

In one embodiment, the polypeptide comprises a first loop whichcomprises the motif G_(r)xWPARG_(r) (SEQ ID No. 3), wherein P isreplaced with azetidine carboxylic acid; and/or R is replaced withN-methyl arginine; and/or R is replaced with homoarginine; and/or R isreplaced with guanidyl-phenylalanine.

In one embodiment, the polypeptide comprises a first loop whichcomprises the motif G_(r)xFPYRG_(r) (SEQ ID No. 6), wherein R isreplaced with N-methyl arginine; and/or R is replaced with homoarginine,and wherein proline is replaced by azetidine carboxylic acid; and/or Ris replaced with guanidyl-phenylalanine.

We have now developed the approaches used in PCT/EP2102/069898, and setforth herein, as applied to the optimization of the second loop. Inaccordance with the present invention, we have identified an additionalprotease recognition site in loop 2, which is located at Histidine 7 inthe 06-34-18 sequence. This residue is in particular recognised byproteases contained in ex vivo human plasma. Moreover, this site is alsorecognised by rat membrane-bound proteases as determined in an in vivorat pharmacokinetic study. Thus, for 06-34-18 to attain a suitablepharmacokinetic stability profile as required for therapeutic purposes,both protease recognition sites (Arg5 in Loop1 and His7 in Loop2) haveto be stabilised such that the peptide is resistant to human plasmaproteases, and the proteolytically aggressive environment encounteredboth in vivo in rat and human.

Accordingly, in a first aspect, the present invention provides a peptideligand specific for human Kallikrein comprising a polypeptide comprisingat least three reactive groups, separated by at least two loopsequences, and a molecular scaffold which forms covalent bonds with thereactive groups of the polypeptide such that at least two polypeptideloops are formed on the molecular scaffold, wherein the loops of thepeptide ligand comprises five amino acids and one loop comprises themotif G_(r) ^(T)/_(L)H^(Q)/_(T)xLG_(r) (SEQ ID No. 212).

In the context of peptide 06-34-18, this loop is the second loop,positioned C-terminal to a first loop.

For example, the motif is G_(r)xHxDLG_(r) (SEQ ID No. 213), whereinG_(r) is a reactive group. For example, the motif G_(r)THxxLG_(r) (SEQID No. 214).

In one embodiment, two adjacent loops of the polypeptide comprise themotif G_(r)x^(W)/_(F)Px^(K)/_(R)G_(r) ^(T)/_(L)H^(Q)/_(T)DLG_(r) (SEQ IDNo. 9).

Examples of such polypeptides are set forth in Table 4, Table 5 or Table6.

In a second aspect, the invention relates to a peptide ligand asdescribed in the first aspect of the invention, wherein removal of His 7in loop 2, or replacement of the histidine side chain with anynon-charged or charged chemical entities, enhances the stability of thepeptide ligand.

For example, proteolytic stability is enhanced by chemical modificationof the peptide bonds N- or C-terminally adjacent to His7. The chemicalmodification is, for example, α-N-alkylation, or modification of thepeptide bond with its reduced amide form.

In one embodiment, proteolytic stability is enhanced by stericobstruction of nearby amino acids to His 7. For example, the stericobstruction results from replacement of position 9 with its D-amino acidenantiomer, or α-N-alkylation.

In another embodiment, proteolytic stability is enhanced by introductionof sterically obstructive amino acids at position 6. For example,proteolytic stability is enhanced by introduction of Cβ-derivatisedamino acids such as phenylglycine or cyclohexylglycine at position 6,which enhance the potency of the peptide ligand sequence towards humanplasma kallikrein and reduce the proteolytic hydrolysis of theC-terminal peptide bond.

The teaching of the present invention may be combined with the teachingof PCT/EP2012/06989, such that the polypeptide according to theinvention can comprise a first loop which comprises the motifG_(r)xWPARG_(r) (SEQ ID No. 3) or G_(r)xFPYRG_(r) (SEQ ID No. 9) and asecond loop comprising the motif G_(r) ^(T)/_(L)H^(Q)/_(T)xLG_(r) (SEQID No. 212).

In one example, in the first loop Pro 3 is replaced with azetidinecarboxylic acid; and/or Arg 5 is replaced with N-methyl arginine; and/orArg 5 is replaced with homoarginine; and/or Arg 5 is replaced withguanidylphenylalanine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Phage selection of bicyclic peptides. (a) Bicyclic peptide phagelibraries. Random amino acids are indicated as ‘X’, alanine as ‘A’ andthe constant three cysteine residues as ‘C’. (b) Format of chemicallysynthesized bicyclic peptide structures having loops of 3, 5 or 6 aminoacids. The structures are generated by linking linear peptides via threecysteine side chains to tris-(bromomethyl)benzene (TBMB). Amino acidsthat vary in the bicyclic peptides are indicated with ‘Xaa’. (c-e)Sequences of bicyclic peptides isolated from library 5×5 (c), library3×3 A (d) and library 3×3 B (e). Similarities in amino acids arehighlighted by shading. ACXXXCXXXCA (SEQ ID No. 177); ACXXXCXXXCA (SEQID No. 178); XCXXXCXXXCX (SEQ ID No. 216); PK100 (SEQ ID No. 33); PK101(SEQ ID No. 34); PK102 (SEQ ID No. 36); PK103 (SEQ ID No. 48); PK104(SEQ ID No. 46); PK105 (SEQ ID No. 35); PK106 (SEQ ID No. 37); PK107(SEQ ID No. 38); PK108 (SEQ ID No. 44). PK 109 (SEQ ID No. 42); PK110(SEQ ID No. 41); PK111 (SEQ ID No. 43); PK112 (SEQ ID No. 47); PK113(SEQ ID No. 45); PK114 (SEQ ID No. 40); PK115 (SEQ ID No. 49); PK116(SEQ ID No. 39); PK117 (SEQ ID No. 209); PK118 (SEQ ID No. 207); PK119(SEQ ID No. 205); PK120 (SEQ ID No. 208); PK121 (SEQ ID No. 206); PK122(SEQ ID No. 203); PK123 (SEQ ID No. 202); PK124 (SEQ ID No. 204); PK125(SEQ ID No. 179); PK126 (SEQ ID No. 180); PK127 (SEQ ID No. 181); PK128(SEQ ID No. 182); PK129 (SEQ ID No. 183); PK130 (SEQ ID No. 184); PK131(SEQ ID No. 185); PK132 (SEQ ID No. 186); PK133 (SEQ ID No. 187); PK134(SEQ ID No. 188); PK135 (SEQ 9 ID No. 189); PK136 (SEQ ID No. 190);PK137 (SEQ ID No. 191); PK138 (SEQ ID No. 192); PK139 (SEQ ID No. 193);PK140 (SEQ ID No. 194); PK141 (SEQ ID No. 195); PK142 (SEQ ID No. 196);PK143 (SEQ ID No. 197)

FIG. 2 Comparison of the surface amino acids of hPK and homologousserine proteases. (a) Structure of hPK (PDB entry 2ANW) with surfacerepresentation. Atoms of amino acids being exposed to the surface andcloser than 4, 8 and 12 Å to benzamidine (in grey) bound to the S1pocket are stained more darkly. (b) Structure of hPK. The side chains ofamino acids that are different in hfXIa are highlighted. (c) Structureof hPK. The side chains of amino acids that are different in rPK arehighlighted.

FIG. 3 Pictorial representation of the method used for determination ofpreferred residues for mutation in polypeptide ligands

FIG. 4 Analysis of amino acid substitutions in peptide 06-34 (Table 4)on the binding of the peptide to plasma Kallikrein at 2 nM. For eachposition, the effect of various mutations at that position is shown, incomparison to the parent sequence.

FIG. 5 Analysis of amino acid substitutions in peptide 06-56 (Table 4)on the binding of the peptide to plasma Kallikrein at 2 nM. For eachposition, the effect of various mutations at that position is shown, incomparison to the parent sequence.

FIG. 6 Analysis of amino acid substitutions in peptide 06-56 (Table 4)on the binding of the peptide to plasma Kallikrein at 10 nM. For eachposition, the effect of various mutations at that position is shown, incomparison to the parent sequence.

FIG. 7 Mass spectrometric output showing the mass spectra ofAc-06-34-18(TMB)-NH2 after exposure to 35% rat plasma, at t0, 1 day, 2days and 3 days (method #1). Mass accuracies vary somewhat due tointerfering ions and low concentrations of fragments; howeveridentification of discrete proteolytic fragments is possible.

FIG. 8 Chemical structures of metabolites M1, M2, M3 ofAc-06-34-18(TMB)-NH2 identified after exposure to rat plasma.

FIG. 9 Chemical structure of the Ac-06-34-18(TMB)-NH2 lead (SEQ ID No.7)

FIG. 10 Enzyme inhibition assay of Kallikrein by theAc-06-34-18(TMB)-NH2 lead and its 1^(st) loop scrambled derivatives. Adramatic reduction in affinity is observed, underlining the importanceof the integrity of the WPAR pharmacophore.

FIG. 11 Chemical structures of arginine and its analogues.

FIG. 12 Chemical structures of Trp and potential hydrophobic analogues

FIG. 13 Chemical structures of Pro and potential constrained analogues

FIG. 14 Comparative Kallikrein inhibition by Aze3, NMeArg5 and doublymodified Ac-06-34-18(TMB)-NH2.

FIG. 15 Chemical structures of Alanine and derivatives thereof

FIG. 16 Comparative Kallikrein inhibition by F2Y4, F2Y4 HR5 and doublymodified Ac-06-34-18(TMB)-NH2.

FIG. 17 Chemical structures of Loop1 modifications in Ac-(06-34-18) thatimpart favorable rat plasma stability and/or potency enhancement (Aze3in place of Pro3, 4GuanPhe in place of Arg5, HArg in place of Arg5,NMe-Arg5 in place of Arg5).

FIG. 18a Wildtype peptide is subjected to human plasma. Note theappearance of fragments with His7-Gln8, Leu6, and Leu6-His7-Gln8removed. Due to the nature of MALDI-TOF, the intensity of the signals isnot quantitative. The possible hydrolysis sites in the sequence areindicated with the arrows in the diagram, with the direction ofsubsequent degradation indicated by the horizontal arrows above thesequence.

FIG. 18b Arg5 N-methylated 06-34-18 is subjected to human plasma. Thispeptide has enhanced stability in rat plasma. In human plasma, however,the peptide is not stable, as exemplified by the appearance of fragmentslacking His7-Gln8, Leu6, and Leu6-His7-Gln8 in Loop2. Due to the natureof MALDI-TOF, the intensity of the signals is not quantitative.

FIG. 19a Chemical Structure of Loop 2 of 06-34-18 (SEQ ID No. 198)

FIG. 19b Summary of structures employed at position 6 in Loop 2. Thenon-standard amino acid acronyms are defined in the Methods and Table 7.

FIG. 19c Summary of structures employed at position 7 in Loop 2. Thenon-standard amino acid acronyms are defined in the Methods and Table 7.

FIG. 19d Summary of structures employed at position 8 in Loop 2. Thenon-standard amino acid acronyms are defined in the Methods and Table 7.

FIG. 20a Structure of N-His Peptoid within Loop 2 of 06-34-18 (SEQ IDNo. 199). A homohistidine side chain was introduced on the Nα, while Cαloses its chiral center.

FIG. 20b Structure of the reduced amide between position 6 and 7 in Loop2 of 06-34-18 (SEQ ID No. 200). The methylene in place of the carbonylremoves the scissile peptide bond.

FIG. 21a Comparative stability of wildtype (06-34-18) at t=0, 21 and 46hrs in human plasma.

FIG. 21b Comparative stability of wildtype (06-34-18) at t=0, 21 and 46hrs in rat plasma.

FIG. 21c Comparative stability of (06-34-18) Phe2 Aze3 Tyr4 HArg5 D-Asp9at t=0, 21 and 46 hrs in human plasma.

FIG. 21d Comparative stability of Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5Ala(ψCH2NH)6 at t=0, 21 and 46 hrs in human plasma.

FIG. 21e Comparative stability of Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5Ala(ψCH2NH)6 (SEQ ID No. 201) at t=0, 21 and 46 hrs in rat plasma.

FIG. 22 Full chemical structure of Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5Ala(ψCH2NH)6 (SEQ ID No. 201). All amino acids are in the L-enantiomericform.

FIG. 23 Rat pharmacokinetic analysis of peptides Ac-(06-34-18) Phe2 Aze3Tyr4 HArg5 Ala(ψCH2NH)6 and Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 D-Asp9compared to the proteolytically stable but pharmacologically inert all-Denantiomer of Ac-(06-34-18). Note the clearance of Ac-(06-34-18) Phe2Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 is closely resembling that of the D aminoacid control peptide “Ac-(06-34-18) all D”. The horizontal dotted linesindicate the limit of quantification for each of the respectivepeptides.

FIG. 24 Human plasma Kallikrein binding of particular motifs atpositions 2, 3, 4 & 5 (with positions 1, 6, 7, 8, 9 &10 fixed to thoseof 06-34-03).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

A peptide ligand, as referred to herein, refers to a peptide covalentlybound to a molecular scaffold. Typically, such peptides comprise two ormore reactive groups which are capable of forming covalent bonds to thescaffold, and a sequence subtended between said reactive groups which isreferred to as the loop sequence, since it forms a loop when the peptideis bound to the scaffold. In the present case, the peptides comprise atleast three reactive groups, and form at least two loops on thescaffold.

The reactive groups are groups capable of forming a covalent bond withthe molecular scaffold. Typically, the reactive groups are present onamino acid side chains on the peptide. Examples are amino-containinggroups such as cysteine, lysine and selenocysteine.

Specificity, in the context herein, refers to the ability of a ligand tobind or otherwise interact with its cognate target to the exclusion ofentities which are similar to the target. For example, specificity canrefer to the ability of a ligand to inhibit the interaction of a humanenzyme, but not a homologous enzyme from a different species. Using theapproach described herein, specificity can be modulated, that isincreased or decreased, so as to make the ligands more or less able tointeract with homologues or paralogues of the intended target.Specificity is not intended to be synonymous with activity, affinity oravidity, and the potency of the action of a ligand on its target (suchas, for example, binding affinity or level of inhibition) are notnecessarily related to its specificity.

Binding activity, as used herein, refers to quantitative bindingmeasurements taken from binding assays, for example as described herein.Therefore, binding activity refers to the amount of peptide ligand whichis bound at a given target concentration.

Multispecificity is the ability to bind to two or more targets.Typically, binding peptides are capable of binding to a single target,such as an epitope in the case of an antibody, due to theirconformational properties. However, peptides can be developed which canbind to two or more targets; dual specific antibodies, for example, asknown in the art as referred to above. In the present invention, thepeptide ligands can be capable of binding to two or more targets and aretherefore be multispecific. For example, they bind to two targets, andare dual specific. The binding may be independent, which would mean thatthe binding sites for the targets on the peptide are not structurallyhindered by the binding of one or other of the targets. In this caseboth targets can be bound independently. More generally it is expectedthat the binding of one target will at least partially impede thebinding of the other.

There is a fundamental difference between a dual specific ligand and aligand with specificity which encompasses two related targets. In thefirst case, the ligand is specific for both targets individually, andinteracts with each in a specific manner. For example, a first loop inthe ligand may bind to a first target, and a second loop to a secondtarget. In the second case, the ligand is non-specific because it doesnot differentiate between the two targets, for example by interactingwith an epitope of the targets which is common to both.

In the context of the present invention, it is possible that a ligandwhich has activity in respect of, for example, a target and anorthologue, could be a bispecific ligand. However, in one embodiment theligand is not bispecific, but has a less precise specificity such thatit binds both the target and one or more orthologues. In general, aligand which has not been selected against both a target and itsorthologue is less likely to be bispecific as a result of modulation ofloop length.

If the ligands are truly bispecific, in one embodiment at least one ofthe target specificities of the ligands will be common amongst theligands selected, and the level of that specificity can be modulated bythe methods disclosed herein. Second or further specificities need notbe shared, and need not be the subject of the procedures set forthherein.

A target is a molecule or part thereof to which the peptide ligands bindor otherwise interact with. Although binding is seen as a prerequisiteto activity of most kinds, and may be an activity in itself, otheractivities are envisaged. Thus, the present invention does not requirethe measurement of binding directly or indirectly.

The molecular scaffold is any molecule which is able to connect thepeptide at multiple points to impart one or more structural features tothe peptide. It is not a cross-linker, in that it does not merelyreplace a disulphide bond; instead, it provides two or more attachmentpoints for the peptide. Suitably, the molecular scaffold comprises atleast three attachment points for the peptide, referred to as scaffoldreactive groups. These groups are capable of reacting to the reactivegroups on the peptide to form a covalent bond. Preferred structures formolecular scaffolds are described below.

Screening for binding activity (or any other desired activity) isconducted according to methods well known in the art, for instance fromphage display technology. For example, targets immobilised to a solidphase can be used to identify and isolate binding members of arepertoire. Screening allows selection of members of a repertoireaccording to desired characteristics.

The term library refers to a mixture of heterogeneous polypeptides ornucleic acids. The library is composed of members, which are notidentical. To this extent, library is synonymous with repertoire.Sequence differences between library members are responsible for thediversity present in the library. The library may take the form of asimple mixture of polypeptides or nucleic acids, or may be in the formof organisms or cells, for example bacteria, viruses, animal or plantcells and the like, transformed with a library of nucleic acids. Undersome conditions, each individual organism or cell contains only one or alimited number of library members.

In one embodiment, the nucleic acids are incorporated into expressionvectors, in order to allow expression of the polypeptides encoded by thenucleic acids. In a preferred aspect, therefore, a library may take theform of a population of host organisms, each organism containing one ormore copies of an expression vector containing a single member of thelibrary in nucleic acid form which can be expressed to produce itscorresponding polypeptide member. Thus, the population of host organismshas the potential to encode a large repertoire of genetically diversepolypeptide variants.

In one embodiment, a library of nucleic acids encodes a repertoire ofpolypeptides. Each nucleic acid member of the library typically has asequence related to one or more other members of the library. By relatedsequence is meant an amino acid sequence having at least 50% identity,for example at least 60% identity, for example at least 70% identity,for example at least 80% identity, for example at least 90% identity,for example at least 95% identity, for example at least 98% identity,for example at least 99% identity to at least one other member of thelibrary. Identity can be judged across a contiguous segment of at least3 amino acids, for example at least 4, 5, 6, 7, 8, 9 or 10 amino acids,for example least 12 amino acids, for example least 14 amino acids, forexample least 16 amino acids, for example least 17 amino acids or thefull length of the reference sequence.

A repertoire is a collection of variants, in this case polypeptidevariants, which differ in their sequence. Typically, the location andnature of the reactive groups will not vary, but the sequences formingthe loops between them can be randomised. Repertoires differ in size,but should be considered to comprise at least 10² members. Repertoiresof 10¹¹ or more members can be constructed.

A set of polypeptide ligands, as used herein, refers to a plurality ofpolypeptide ligands which can be subjected to selection in the methodsdescribed. Potentially, a set can be a repertoire, but it may also be asmall collection of polypeptides, from at least 2 up to 10, 20, 50, 100or more.

A group of polypeptide ligands, as used herein, refers to two or moreligands. In one embodiment, a group of ligands comprises only ligandswhich share at least one target specificity. Typically, a group willconsist of from at least 2, 3, 4, 5, 6, 7, 8, 9 or 10, 20, 50, 100 ormore ligands. In one embodiment, a group consists of 2 ligands.

(A) Construction of Peptide Ligands

(i) Molecular Scaffold

Molecular scaffolds are described in, for example, WO2009098450 andreferences cited therein, particularly WO2004077062 and WO2006078161.

As noted in the foregoing documents, the molecular scaffold may be asmall molecule, such as a small organic molecule.

In one embodiment the molecular scaffold may be, or may be based on,natural monomers such as nucleosides, sugars, or steroids. For examplethe molecular scaffold may comprise a short polymer of such entities,such as a dimer or a trimer.

In one embodiment the molecular scaffold is a compound of knowntoxicity, for example of low toxicity. Examples of suitable compoundsinclude cholesterols, nucleotides, steroids, or existing drugs such astamazepam.

In one embodiment the molecular scaffold may be a macromolecule. In oneembodiment the molecular scaffold is a macromolecule composed of aminoacids, nucleotides or carbohydrates.

In one embodiment the molecular scaffold comprises reactive groups thatare capable of reacting with functional group(s) of the polypeptide toform covalent bonds.

The molecular scaffold may comprise chemical groups as amines, thiols,alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters,alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkylhalides and acyl halides.

In one embodiment, the molecular scaffold may comprise or may consist oftris(bromomethyl)benzene, especially 1,3,5-Tris(bromomethyl)benzene(‘TBMB’), or a derivative thereof.

In one embodiment, the molecular scaffold is2,4,6-Tris(bromomethyl)mesitylene. It is similar to1,3,5-Tris(bromomethyl)benzene but contains additionally three methylgroups attached to the benzene ring. This has the advantage that theadditional methyl groups may form further contacts with the polypeptideand hence add additional structural constraint.

The molecular scaffold of the invention contains chemical groups thatallow functional groups of the polypeptide of the encoded library of theinvention to form covalent links with the molecular scaffold. Saidchemical groups are selected from a wide range of functionalitiesincluding amines, thiols, alcohols, ketones, aldehydes, nitriles,carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides,maleimides, azides, alkyl halides and acyl halides.

(ii) Polypeptide

The reactive groups of the polypeptides can be provided by side chainsof natural or non-natural amino acids. The reactive groups of thepolypeptides can be selected from thiol groups, amino groups, carboxylgroups, guanidinium groups, phenolic groups or hydroxyl groups. Thereactive groups of the polypeptides can be selected from azide,keto-carbonyl, alkyne, vinyl, or aryl halide groups. The reactive groupsof the polypeptides for linking to a molecular scaffold can be the aminoor carboxy termini of the polypeptide.

In some embodiments each of the reactive groups of the polypeptide forlinking to a molecular scaffold are of the same type. For example, eachreactive group may be a cysteine residue. Further details are providedin WO2009098450.

In some embodiments the reactive groups for linking to a molecularscaffold may comprise two or more different types, or may comprise threeor more different types. For example, the reactive groups may comprisetwo cysteine residues and one lysine residue, or may comprise onecysteine residue, one lysine residue and one N-terminal amine.

Cysteine can be employed because it has the advantage that itsreactivity is most different from all other amino acids. Scaffoldreactive groups that could be used on the molecular scaffold to reactwith thiol groups of cysteines are alkyl halides (or also namedhalogenoalkanes or haloalkanes). Examples are bromomethylbenzene (thescaffold reactive group exemplified by TBMB) or iodoacetamide. Otherscaffold reactive groups that are used to couple selectively compoundsto cysteines in proteins are maleimides. Examples of maleimides whichmay be used as molecular scaffolds in the invention include:tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene,tris-(maleimido)benzene. Selenocysteine is also a natural amino acidwhich has a similar reactivity to cysteine and can be used for the samereactions. Thus, wherever cysteine is mentioned, it is typicallyacceptable to substitute selenocysteine unless the context suggestsotherwise.

Lysines (and primary amines of the N-terminus of peptides) are alsosuited as reactive groups to modify peptides on phage by linking to amolecular scaffold. However, they are more abundant in phage proteinsthan cysteines and there is a higher risk that phage particles mightbecome cross-linked or that they might lose their infectivity.Nevertheless, it has been found that lysines are especially useful inintramolecular reactions (e.g. when a molecular scaffold is alreadylinked to the phage peptide) to form a second or consecutive linkagewith the molecular scaffold. In this case the molecular scaffold reactspreferentially with lysines of the displayed peptide (in particularlysines that are in close proximity). Scaffold reactive groups thatreact selectively with primary amines are succinimides, aldehydes oralkyl halides. In the bromomethyl group that is used in a number of theaccompanying examples, the electrons of the benzene ring can stabilizethe cationic transition state. This particular aryl halide is therefore100-1000 times more reactive than alkyl halides. Examples ofsuccinimides for use as molecular scaffold include tris-(succinimidylaminotriacetate), 1,3,5-Benzenetriacetic acid. Examples of aldehydes foruse as molecular scaffold include Triformylmethane. Examples of alkylhalides for use as molecular scaffold include1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene, 1,3,5-Tris(bromomethyl)benzene, 1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene.

The amino acids with reactive groups for linking to a molecular scaffoldmay be located at any suitable positions within the polypeptide. Inorder to influence the particular structures or loops created, thepositions of the amino acids having the reactive groups may be varied bythe skilled operator, e.g. by manipulation of the nucleic acid encodingthe polypeptide in order to mutate the polypeptide produced. By suchmeans, loop length can be manipulated in accordance with the presentteaching.

For example, the polypeptide can comprise the sequenceAC(X)_(n)C(X)_(m)CG (SEQ ID No. 10), wherein X stands for a randomnatural amino acid, A for alanine, C for cysteine and G for glycine andn and m, which may be the same or different, are numbers between 3 and6.

(iii) Reactive Groups of the Polypeptide

The molecular scaffold of the invention may be bonded to the polypeptidevia functional or reactive groups on the polypeptide. These aretypically formed from the side chains of particular amino acids found inthe polypeptide polymer. Such reactive groups may be a cysteine sidechain, a lysine side chain, or an N-terminal amine group or any othersuitable reactive group. Again, details may be found in WO2009098450.

Examples of reactive groups of natural amino acids are the thiol groupof cysteine, the amino group of lysine, the carboxyl group of aspartateor glutamate, the guanidinium group of arginine, the phenolic group oftyrosine or the hydroxyl group of serine. Non-natural amino acids canprovide a wide range of reactive groups including an azide, aketo-carbonyl, an alkyne, a vinyl, or an aryl halide group. The aminoand carboxyl group of the termini of the polypeptide can also serve asreactive groups to form covalent bonds to a molecular scaffold/molecularcore.

The polypeptides of the invention contain at least three reactivegroups. Said polypeptides can also contain four or more reactive groups.The more reactive groups are used, the more loops can be formed in themolecular scaffold.

In a preferred embodiment, polypeptides with three reactive groups aregenerated. Reaction of said polypeptides with a molecularscaffold/molecular core having a three-fold rotational symmetrygenerates a single product isomer. The generation of a single productisomer is favourable for several reasons. The nucleic acids of thecompound libraries encode only the primary sequences of the polypeptidebut not the isomeric state of the molecules that are formed uponreaction of the polypeptide with the molecular core. If only one productisomer can be formed, the assignment of the nucleic acid to the productisomer is clearly defined. If multiple product isomers are formed, thenucleic acid cannot give information about the nature of the productisomer that was isolated in a screening or selection process. Theformation of a single product isomer is also advantageous if a specificmember of a library of the invention is synthesized. In this case, thechemical reaction of the polypeptide with the molecular scaffold yieldsa single product isomer rather than a mixture of isomers.

In another embodiment of the invention, polypeptides with four reactivegroups are generated. Reaction of said polypeptides with a molecularscaffold/molecular core having a tetrahedral symmetry generates twoproduct isomers. Even though the two different product isomers areencoded by one and the same nucleic acid, the isomeric nature of theisolated isomer can be determined by chemically synthesizing bothisomers, separating the two isomers and testing both isomers for bindingto a target ligand.

In one embodiment of the invention, at least one of the reactive groupsof the polypeptides is orthogonal to the remaining reactive groups. Theuse of orthogonal reactive groups allows the directing of saidorthogonal reactive groups to specific sites of the molecular core.Linking strategies involving orthogonal reactive groups may be used tolimit the number of product isomers formed. In other words, by choosingdistinct or different reactive groups for one or more of the at leastthree bonds to those chosen for the remainder of the at least threebonds, a particular order of bonding or directing of specific reactivegroups of the polypeptide to specific positions on the molecularscaffold may be usefully achieved.

In another embodiment, the reactive groups of the polypeptide of theinvention are reacted with molecular linkers wherein said linkers arecapable to react with a molecular scaffold so that the linker willintervene between the molecular scaffold and the polypeptide in thefinal bonded state.

In some embodiments, amino acids of the members of the libraries or setsof polypeptides can be replaced by any natural or non-natural aminoacid. Excluded from these exchangeable amino acids are the onesharbouring functional groups for cross-linking the polypeptides to amolecular core, such that the loop sequences alone are exchangeable. Theexchangeable polypeptide sequences have either random sequences,constant sequences or sequences with random and constant amino acids.The amino acids with reactive groups are either located in definedpositions within the polypeptide, since the position of these aminoacids determines loop size.

In one embodiment, an polypeptide with three reactive groups has thesequence (X)_(l)Y(X)_(m)Y(X)_(n)Y(X)_(o) (SEQ ID No. 215), wherein Yrepresents an amino acid with a reactive group, X represents a randomamino acid, m and n are numbers between 3 and 6 defining the length ofintervening polypeptide segments, which may be the same or different,and l and o are numbers between 0 and 20 defining the length of flankingpolypeptide segments.

Alternatives to thiol-mediated conjugations can be used to attach themolecular scaffold to the peptide via covalent interactions.Alternatively these techniques may be used in modification or attachmentof further moieties (such as small molecules of interest which aredistinct from the molecular scaffold) to the polypeptide after they havebeen selected or isolated according to the present invention—in thisembodiment then clearly the attachment need not be covalent and mayembrace non-covalent attachment. These methods may be used instead of(or in combination with) the thiol mediated methods by producing phagethat display proteins and peptides bearing unnatural amino acids withthe requisite chemical reactive groups, in combination small moleculesthat bear the complementary reactive group, or by incorporating theunnatural amino acids into a chemically or recombinantly synthesisedpolypeptide when the molecule is being made after theselection/isolation phase. Further details can be found in WO2009098450or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.

(iv) Combination of Loops to Form Multispecific Molecules

Loops from peptide ligands, or repertoires of peptide ligands, areadvantageously combined by sequencing and de novo synthesis of apolypeptide incorporating the combined loops. Alternatively, nucleicacids encoding such polypeptides can be synthesised.

Where repertoires are to be combined, particularly single looprepertoires, the nucleic acids encoding the repertoires areadvantageously digested and re-ligated, to form a novel repertoirehaving different combinations of loops from the constituent repertoires.Phage vectors can include polylinkers and other sites for restrictionenzymes which can provide unique points for cutting and relegation thevectors, to create the desired multispecific peptide ligands. Methodsfor manipulating phage libraries are well known in respect ofantibodies, and can be applied in the present case also.

(v) Attachment of Effector Groups and Functional Groups

Effector and/or functional groups can be attached, for example, to the Nor C termini of the polypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragmentsthereof. For instance, an effector group can include an antibody lightchain constant region (CL), an antibody CH1 heavy chain domain, anantibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, orany combination thereof, in addition to the one or more constant regiondomains. An effector group may also comprise a hinge region of anantibody (such a region normally being found between the CH1 and CH2domains of an IgG molecule).

In a further preferred embodiment of this aspect of the invention, aneffector group according to the present invention is an Fc region of anIgG molecule. Advantageously, a peptide ligand-effector group accordingto the present invention comprises or consists of a peptide ligand Fcfusion having a tβ half-life of a day or more, two days or more, 3 daysor more, 4 days or more, 5 days or more, 6 days or more or 7 days ormore. Most advantageously, the peptide ligand according to the presentinvention comprises or consists of a peptide ligand Fc fusion having atβ half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactivegroups for the attachment of other entities, functional groups which aiduptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptidesagainst intracellular targets to be effective. Targets that can beaccessed by peptides with the ability to penetrate into cells includetranscription factors, intracellular signalling molecules such astyrosine kinases and molecules involved in the apoptotic pathway.Functional groups which enable the penetration of cells include peptidesor chemical groups which have been added either to the peptide or themolecular scaffold. Peptides such as those derived from such as VP22,HIV-Tat, a homeobox protein of Drosophila (Antennapedia), e.g. asdescribed in Chen and Harrison, Biochemical Society Transactions (2007)Volume 35, part 4, p 821 “Cell-penetrating peptides in drug development:enabling intracellular targets” and “Intracellular delivery of largemolecules and small peptides by cell penetrating peptides” by Gupta etal. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examplesof short peptides which have been shown to be efficient at translocationthrough plasma membranes include the 16 amino acid penetratin peptidefrom Drosophila Antennapedia protein (Derossi et al (1994) J Biol. Chem.Volume 269 p 10444 “The third helix of the Antennapedia homeodomaintranslocates through biological membranes”), the 18 amino acid ‘modelamphipathic peptide’ (Oehlke et al (1998) Biochim Biophys Acts Volume1414 p 127 “Cellular uptake of an alpha-helical amphipathic modelpeptide with the potential to deliver polar compounds into the cellinterior non-endocytically”) and arginine rich regions of the HIV TATprotein. Non peptidic approaches include the use of small moleculemimics or SMOCs that can be easily attached to biomolecules (Okuyama etal (2007) Nature Methods Volume 4 p 153 ‘Small-molecule mimics of ana-helix for efficient transport of proteins into cells’. Other chemicalstrategies to add guanidinium groups to molecules also enhance cellpenetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p 13585“Guanidinylated Neomcyin Delivers Large Bioactive Cargo into cellsthrough a heparin Sulphate Dependent Pathway”). Small molecular weightmolecules such as steroids may be added to the molecular scaffold toenhance uptake into cells.

One class of functional groups which may be attached to peptide ligandsincludes antibodies and binding fragments thereof, such as Fab, Fv orsingle domain fragments. In particular, antibodies which bind toproteins capable of increasing the half life of the peptide ligand invivo may be used.

RGD peptides, which bind to integrins which are present on many cells,may also be incorporated.

In one embodiment, a peptide ligand-effector group according to theinvention has a tβ half-life selected from the group consisting of: 12hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 daysor more, 5 days or more, 6 days or more, 7 days or more, 8 days or more,9 days or more, 10 days or more, 11 days or more, 12 days or more, 13days or more, 14 days or more, 15 days or more or 20 days or more.Advantageously a peptide ligand-effector group or composition accordingto the invention will have a tβ half life in the range 12 to 60 hours.In a further embodiment, it will have a t half-life of a day or more. Ina further embodiment still, it will be in the range 12 to 26 hours.

Functional groups include drugs, such as cytotoxic agents for cancertherapy. These include Alkylating agents such as Cisplatin andcarboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, ifosfamide; Anti-metabolites including purine analogsazathioprine and mercaptopurine)) or pyrimidine analogs; plant alkaloidsand terpenoids including vinca alkaloids such as Vincristine,Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and itsderivatives etoposide and teniposide; Taxanes, including paclitaxel,originally known as Taxol; topoisomerase inhibitors includingcamptothecins: irinotecan and topotecan, and type II inhibitorsincluding amsacrine, etoposide, etoposide phosphate, and teniposide.Further agents can include Antitumour antibiotics which include theimmunosuppressant dactinomycin (which is used in kidneytransplantations), doxorubicin, epirubicin, bleomycin and others.

Possible effector groups also include enzymes, for instance such ascarboxypeptidase G2 for use in enzyme/prodrug therapy, where the peptideligand replaces antibodies in ADEPT.

(vi) Synthesis

It should be noted that once a polypeptide of interest is isolated oridentified according to the present invention, then its subsequentsynthesis may be simplified wherever possible. Thus, groups or sets ofpolypeptides need not be produced by recombinant DNA techniques. Forexample, the sequence of polypeptides of interest may be determined, andthey may be manufactured synthetically by standard techniques followedby reaction with a molecular scaffold in vitro. When this is performed,standard chemistry may be used since there is no longer any need topreserve the functionality or integrity of the genetically encodedcarrier particle, such as phage. This enables the rapid large scalepreparation of soluble material for further downstream experiments orvalidation. In this regard, large scale preparation of the candidates orleads identified by the methods of the present invention could beaccomplished using conventional chemistry such as that disclosed inTimmerman et al.

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. In one embodiment, thesesteps are carried out on the end product polypeptide/conjugate made bychemical synthesis, rather than on the phage.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex e.g. after theinitial isolation/identification step.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus or within the loops using orthogonallyprotected lysines (and analogues) using standard solid phase or solutionphase chemistry. Standard protein chemistry may be used to introduce anactivatable N- or C-terminus. Alternatively additions may be made byfragment condensation or native chemical ligation e.g. as described in(Dawson P E, Muir T W, Clark-Lewis I, Kent, S B H. 1994. Synthesis ofProteins by Native Chemical Ligation. Science 266:776-779), or byenzymes, for example using subtiligase as described in (Subtiligase: atool for semisynthesis of proteins Chang T K, Jackson D Y, Burnier J P,Wells J A Proc Natl Acad Sci USA. 1994 Dec. 20; 91(26):12544-8 or inBioorganic & Medicinal Chemistry Letters Tags for labelling proteinN-termini with subtiligase for proteomics Volume 18, Issue 22, 15 Nov.2008, Pages 6000-6003 Tags for labeling protein N-termini withsubtiligase for proteomics; Hikari A. I. Yoshihara, Sami Mahrus andJames A. Wells).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptide to dissociate from each otheronce within the reducing environment of the cell. In this case, themolecular scaffold (eg. TBMB) could be added during the chemicalsynthesis of the first peptide so as to react with the three cysteinegroups; a further cysteine could then be appended to the N-terminus ofthe first peptide, so that this cysteine only reacted with a freecysteine of the second peptide.

Similar techniques apply equally to the synthesis/coupling of twobicyclic and bispecific macrocycles, potentially creating atetraspecific molecule.

Furthermore, addition of other functional groups or effector groups maybe accomplished in the same manner, using appropriate chemistry,coupling at the N- or C-termini or via side chains. In one embodiment,the coupling is conducted in such a manner that it does not block theactivity of either entity.

(vii) Peptide Modification

To develop the bicyclic peptides (Bicycles; peptides conjugated tomolecular scaffolds) into a suitable drug-like molecule, whether that befor injection, inhalation, nasal, ocular, oral or topicaladministration, a number of properties need considered. The following atleast need to be designed into a given lead Bicycle:

-   -   protease stability, whether this concerns Bicycle stability to        plasma proteases, epithelial (“membrane-anchored”) proteases,        gastric and intestinal proteases, lung surface proteases,        intracellular proteases and the like. Protease stability should        be maintained between different species such that a Bicycle lead        candidate can be developed in animal models as well as        administered with confidence to humans.    -   replacement of oxidation-sensitive residues, such as tryptophan        and methionine with oxidation-resistant analogues in order to        improve the pharmaceutical stability profile of the molecule    -   a desirable solubility profile, which is a function of the        proportion of charged and hydrophilic versus hydrophobic        residues, which is important for formulation and absorption        purposes    -   correct balance of charged versus hydrophobic residues, as        hydrophobic residues influence the degree of plasma protein        binding and thus the concentration of the free available        fraction in plasma, while charged residues (in particular        arginines) may influence the interaction of the peptide with the        phospholipid membranes on cell surfaces. The two in combination        may influence half-life, volume of distribution and exposure of        the peptide drug, and can be tailored according to the clinical        endpoint. In addition, the correct combination and number of        charged versus hydrophobic residues may reduce irritation at the        injection site (were the peptide drug administered        subcutaneously).    -   a tailored half-life, depending on the clinical indication and        treatment regimen. It may be prudent to develop an unmodified        molecule for short exposure in an acute illness management        setting, or develop a bicyclic peptide with chemical        modifications that enhance the plasma half-life, and hence be        optimal for the management of more chronic disease states.

Approaches to stabilise therapeutic peptide candidates againstproteolytic degradation are numerous, and overlap with thepeptidomimetics field (for reviews see Gentilucci et al, Curr.Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr.Medicinal Chem (2009), 16, 4399-418).

These include

-   -   Cyclisation of peptide    -   N- and C-terminal capping, usually N-terminal acetylation and        C-terminal amidation.    -   Alanine scans, to reveal and potentially remove the proteolytic        attack site(s).    -   D-amino acid replacement, to probe the steric requirements of        the amino acid side chain, to increase proteolytic stability by        steric hindrance and by a propensity of D-amino acids to        stabilise β-turn conformations (Tugyi et al (2005) PNAS, 102(2),        413-418).    -   N-methyl/N-alkyl amino acid replacement, to impart proteolytic        protection by direct modification of the scissile amide bond        (Fiacco et al, Chembiochem. (2008), 9(14), 2200-3).        N-methylation also has strong effect on the torsional angles of        the peptide bond, and is believed to aid in cell penetration &        oral availability (Biron et al (2008), Angew. Chem. Int. Ed.,        47, 2595-99)    -   Incorporation of non-natural amino acids, i.e. by employing        -   Isosteric/isoelectronic side chains that are not recognised            by proteases, yet have no effect on target potency        -   Constrained amino acid side chains, such that proteolytic            hydrolysis of the nearby peptide bond is conformationally            and sterically impeded. In particular, these concern proline            analogues, bulky sidechains, Cα-disubstituted derivatives            (where the simplest derivative is Aib, H₂N—C(CH₃)₂—COOH),            and cyclo amino acids, a simple derivative being            amino-cyclopropylcarboxylic acid).    -   Peptide bond surrogates, and examples include        -   N-alkylation (see above, i.e. CO—NR)        -   Reduced peptide bonds (CH₂—NH—)        -   Peptoids (N-alkyl amino acids, NR—CH₂—CO)        -   Thio-amides (CS—NH)        -   Azapeptides (CO—NH—NR)        -   Trans-alkene (RHC═C—)        -   Retro-inverso (NH—CO)        -   Urea surrogates (NH—CO—NHR)    -   Peptide backbone length modulation        -   i.e. β^(2/3)-amino acids, (NH—CR—CH₂—CO, NH—CH₂—CHR—CO),    -   Substitutions on the alpha-carbon on amino acids, which        constrains backbone conformations, the simplest derivative being        Aminoisobutyric acid (Aib).

It should be explicitly noted that some of these modifications may alsoserve to deliberately improve the potency of the peptide against thetarget, or, for example to identify potent substitutes for theoxidation-sensitive amino acids (Trp and Met). It should also be notedthat the Bicycle lead Ac-06-34-18(TMB)-NH2 already harbours twomodifications that impart resistance to proteolytic degradation, thesebeing N/C-terminal capping, and (bi)cyclisation.

(B) Repertoires, Sets and Groups of Polypeptide Ligands

(i) Construction of Libraries

Libraries intended for selection may be constructed using techniquesknown in the art, for example as set forth in WO2004/077062, orbiological systems, including phage vector systems as described herein.Other vector systems are known in the art, and include other phage (forinstance, phage lambda), bacterial plasmid expression vectors,eukaryotic cell-based expression vectors, including yeast vectors, andthe like. For example, see WO2009098450 or Heinis, et al., Nat Chem Biol2009, 5 (7), 502-7.

Non-biological systems such as those set forth in WO2004/077062 arebased on conventional chemical screening approaches. They are simple,but lack the power of biological systems since it is impossible, or atleast impracticably onerous, to screen large libraries of peptideligands. However, they are useful where, for instance, only a smallnumber of peptide ligands needs to be screened. Screening by suchindividual assays, however, may be time-consuming and the number ofunique molecules that can be tested for binding to a specific targetgenerally does not exceed 10⁶ chemical entities.

In contrast, biological screening or selection methods generally allowthe sampling of a much larger number of different molecules. Thusbiological methods can be used in application of the invention. Inbiological procedures, molecules are assayed in a single reaction vesseland the ones with favourable properties (i.e. binding) are physicallyseparated from inactive molecules. Selection strategies are availablethat allow to generate and assay simultaneously more than 10¹³individual compounds. Examples for powerful affinity selectiontechniques are phage display, ribosome display, mRNA display, yeastdisplay, bacterial display or RNA/DNA aptamer methods. These biologicalin vitro selection methods have in common that ligand repertoires areencoded by DNA or RNA. They allow the propagation and the identificationof selected ligands by sequencing. Phage display technology has forexample been used for the isolation of antibodies with very high bindingaffinities to virtually any target.

When using a biological system, once a vector system is chosen and oneor more nucleic acid sequences encoding polypeptides of interest arecloned into the library vector, one may generate diversity within thecloned molecules by undertaking mutagenesis prior to expression;alternatively, the encoded proteins may be expressed and selected beforemutagenesis and additional rounds of selection are performed.

Mutagenesis of nucleic acid sequences encoding structurally optimisedpolypeptides is carried out by standard molecular methods. Of particularuse is the polymerase chain reaction, or PCR, (Mullis and Faloona (1987)Methods Enzymol., 155: 335, herein incorporated by reference). PCR,which uses multiple cycles of DNA replication catalysed by athermostable, DNA-dependent DNA polymerase to amplify the targetsequence of interest, is well known in the art. The construction ofvarious antibody libraries has been discussed in Winter et al. (1994)Ann. Rev. Immunology 12, 433-55, and references cited therein.

Alternatively, given the short chain lengths of the polypeptidesaccording to the invention, the variants can be synthesised de novo andinserted into suitable expression vectors. Peptide synthesis can becarried out by standard techniques known in the art, as described above.Automated peptide synthesisers are widely available, such as the AppliedBiosystems ABI 433 (Applied Biosystems, Foster City, Calif., USA)

(ii) Genetically Encoded Diversity

In one embodiment, the polypeptides of interest are genetically encoded.This offers the advantage of enhanced diversity together with ease ofhandling. An example of a genetically polypeptide library is a mRNAdisplay library. Another example is a replicable genetic display package(rgdp) library such as a phage display library. In one embodiment, thepolypeptides of interest are genetically encoded as a phage displaylibrary.

Thus, in one embodiment the complex of the invention comprises areplicable genetic display package (rgdp) such as a phage particle. Inthese embodiments, the nucleic acid can be comprised by the phagegenome. In these embodiments, the polypeptide can be comprised by thephage coat.

In some embodiments, the invention may be used to produce a geneticallyencoded combinatorial library of polypeptides which are generated bytranslating a number of nucleic acids into corresponding polypeptidesand linking molecules of said molecular scaffold to said polypeptides.

The genetically encoded combinatorial library of polypeptides may begenerated by phage display, yeast display, ribosome display, bacterialdisplay or mRNA display.

Techniques and methodology for performing phage display can be found inWO2009098450.

In one embodiment, screening may be performed by contacting a library,set or group of polypeptide ligands with a target and isolating one ormore member(s) that bind to said target.

In another embodiment, individual members of said library, set or groupare contacted with a target in a screen and members of said library thatbind to said target are identified.

In another embodiment, members of said library, set or group aresimultaneously contacted with a target and members that bind to saidtarget are selected.

The target(s) may be a peptide, a protein, a polysaccharide, a lipid, aDNA or a RNA.

The target may be a receptor, a receptor ligand, an enzyme, a hormone ora cytokine.

The target may be a prokaryotic protein, a eukaryotic protein, or anarcheal protein. More specifically the target ligand may be a mammalianprotein or an insect protein or a bacterial protein or a fungal proteinor a viral protein.

The target ligand may be an enzyme, such as a protease.

It should be noted that the invention also embraces polypeptide ligandsisolated from a screen according to the invention. In one embodiment thescreening method(s) of the invention further comprise the step of:manufacturing a quantity of the polypeptide isolated as capable ofbinding to said targets.

The invention also relates to peptide ligands having more than twoloops. For example, tricyclic polypeptides joined to a molecularscaffold can be created by joining the N- and C-termini of a bicyclicpolypeptide joined to a molecular scaffold according to the presentinvention. In this manner, the joined N and C termini create a thirdloop, making a tricyclic polypeptide. This embodiment need not becarried out on phage, but can be carried out on a polypeptide-molecularscaffold conjugate as described herein. Joining the N- and C-termini isa matter of routine peptide chemistry. In case any guidance is needed,the C-terminus may be activated and/or the N- and C-termini may beextended for example to add a cysteine to each end and then join them bydisulphide bonding. Alternatively the joining may be accomplished by useof a linker region incorporated into the N/C termini. Alternatively theN and C termini may be joined by a conventional peptide bond.Alternatively any other suitable means for joining the N and C terminimay be employed, for example N—C-cyclization could be done by standardtechniques, for example as disclosed in Linde et al. Peptide Science 90,671-682 (2008) “Structure-activity relationship and metabolic stabilitystudies of backbone cyclization and N-methylation of melanocortinpeptides”, or as in Hess et al. J. Med. Chem. 51, 1026-1034 (2008)“backbone cyclic peptidomimetic melanocortin-4 receptor agonist as anovel orally administered drug lead for treating obesity”. One advantageof such tricyclic molecules is the avoidance of proteolytic degradationof the free ends, in particular by exoprotease action. Another advantageof a tricyclic polypeptide of this nature is that the third loop may beutilised for generally applicable functions such as BSA binding, cellentry or transportation effects, tagging or any other such use. It willbe noted that this third loop will not typically be available forselection (because it is not produced on the phage but only on thepolypeptide-molecular scaffold conjugate) and so its use for other suchbiological functions still advantageously leaves both loops 1 and 2 forselection/creation of specificity.

(iii) Phage Purification

Any suitable means for purification of the phage may be used. Standardtechniques may be applied in the present invention. For example, phagemay be purified by filtration or by precipitation such as PEGprecipitation; phage particles may be produced and purified bypolyethylene-glycol (PEG) precipitation as described previously. Detailscan be found in WO2009098450.

In case further guidance is needed, reference is made to Jespers et al(Protein Engineering Design and Selection 2004 17(10):709-713. Selectionof optical biosensors from chemisynthetic antibody libraries.) In oneembodiment phage may be purified as taught therein. The text of thispublication is specifically incorporated herein by reference for themethod of phage purification; in particular reference is made to thematerials and methods section starting part way down the right-column atpage 709 of Jespers et al.

Moreover, the phage may be purified as published by Marks et al J. Mol.Biol vol 222 pp 581-597, which is specifically incorporated herein byreference for the particular description of how the phageproduction/purification is carried out.

(iv) Reaction Chemistry

The present invention makes use of chemical conditions for themodification of polypeptides which advantageously retain the functionand integrity of the genetically encoded element of the product.Specifically, when the genetically encoded element is a polypeptidedisplayed on the surface of a phage encoding it, the chemistryadvantageously does not compromise the biological integrity of thephage. In general, conditions are set out in WO2009098450.

(C) Use of Polypeptide Ligands According to the Invention

Polypeptide ligands selected according to the method of the presentinvention may be employed in in vivo therapeutic and prophylacticapplications, in vitro and in vivo diagnostic applications, in vitroassay and reagent applications, and the like. Ligands having selectedlevels of specificity are useful in applications which involve testingin non-human animals, where cross-reactivity is desirable, or indiagnostic applications, where cross-reactivity with homologues orparalogues needs to be carefully controlled. In some applications, suchas vaccine applications, the ability to elicit an immune response topredetermined ranges of antigens can be exploited to tailor a vaccine tospecific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity arepreferred for administration to a mammal, and 98 to 99% or morehomogeneity is most preferred for pharmaceutical uses, especially whenthe mammal is a human. Once purified, partially or to homogeneity asdesired, the selected polypeptides may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

The peptide ligands of the present invention will typically find use inpreventing, suppressing or treating inflammatory states, allergichypersensitivity, cancer, bacterial or viral infection, and autoimmunedisorders (which include, but are not limited to, Type I diabetes,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,Crohn's disease and myasthenia gravis).

In the instant application, the term “prevention” involvesadministration of the protective composition prior to the induction ofthe disease. “Suppression” refers to administration of the compositionafter an inductive event, but prior to the clinical appearance of thedisease. “Treatment” involves administration of the protectivecomposition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels.

Methods for the testing of systemic lupus erythematosus (SLE) insusceptible mice are known in the art (Knight et al. (1978) J Exp. Med.,147: 1653; Reinersten et al. (1978) New Eng. J: Med., 299: 515).Myasthenia Gravis (MG) is tested in SJL/J female mice by inducing thedisease with soluble AchR protein from another species (Lindstrom et al.(1988) Adv. Inzn7unol., 42: 233). Arthritis is induced in a susceptiblestrain of mice by injection of Type II collagen (Stuart et al. (1984)Ann. Rev. Immunol., 42: 233). A model by which adjuvant arthritis isinduced in susceptible rats by injection of mycobacterial heat shockprotein has been described (Van Eden et al. (1988) Nature, 331: 171).Thyroiditis is induced in mice by administration of thyroglobulin asdescribed (Maron et al. (1980) J. Exp. Med., 152: 1115). Insulindependent diabetes mellitus (IDDM) occurs naturally or can be induced incertain strains of mice such as those described by Kanasawa et al.(1984) Diabetologia, 27: 113. EAE in mouse and rat serves as a model forMS in human. In this model, the demyelinating disease is induced byadministration of myelin basic protein (see Paterson (1986) Textbook ofImmunopathology, Mischer et al., eds., Grune and Stratton, New York, pp.179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al.(1987) J; Immunol., 138: 179).

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate carriers. Typically, thesecarriers include aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, any including saline and/or buffered media. Parenteralvehicles include sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride and lactated Ringer's. Suitablephysiologically-acceptable adjuvants, if necessary to keep a polypeptidecomplex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cyclosporine, methotrexate, adriamycin or cisplatinum,and immunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the selectedantibodies, receptors or binding proteins thereof of the presentinvention, or even combinations of selected polypeptides according tothe present invention having different specificities, such aspolypeptides selected using different target ligands, whether or notthey are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected antibodies, receptors or binding proteinsthereof of the invention can be administered to any patient inaccordance with standard techniques. The administration can be by anyappropriate mode, including parenterally, intravenously,intramuscularly, intraperitoneally, transdermally, via the pulmonaryroute, or also, appropriately, by direct infusion with a catheter. Thedosage and frequency of administration will depend on the age, sex andcondition of the patient, concurrent administration of other drugs,counterindications and other parameters to be taken into account by theclinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that use levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the selectedrepertoires of polypeptides described herein may be usedextracorporeally or in vitro selectively to kill, deplete or otherwiseeffectively remove a target cell population from a heterogeneouscollection of cells. Blood from a mammal may be combinedextracorporeally with the selected peptide ligands whereby the undesiredcells are killed or otherwise removed from the blood for return to themammal in accordance with standard techniques.

According to a further aspect of the invention, there is provided amethod of preventing, suppressing or treating inflammatory states,allergic hypersensitivity, cancer, bacterial or viral infection,ophthalmic disorders and autoimmune disorders which comprisesadministering to a patient in need thereof a peptide ligand as definedherein.

Examples of suitable “ophthalmic disorders” (including exudative and/orinflammatory ophthalmic disorders, disorders related to impaired retinalvessel permeability and/or integrity, disorders related to retinalmicrovessel rupture leading to focal hemorrhages, back of the eyediseases, retinal diseases and front of the eye diseases) include butare not limited to: age related macular degeneration (ARMD), exudativemacular degeneration (also known as “wet” or neovascular age-relatedmacular degeneration (wet-AMD), macular oedema, aged disciform maculardegeneration, cystoid macular oedema, palpebral oedema, retinal oedema,diabetic retinopathy, acute macular neuroretinopathy, central serouschorioretinopathy, chorioretinopathy, choroidal neovascularization,neovascular maculopathy, neovascular glaucoma, obstructive arterial andvenous retinopathies (e.g. retinal venous occlusion or retinal arterialocclusion), central retinal vein occlusion, disseminated intravascularcoagulopathy, branch retinal vein occlusion, hypertensive funduschanges, ocular ischemic syndrome, retinal arterial microaneurysms,Coat's disease, parafoveal telangiectasis, hemi-retinal vein occlusion,papillophlebitis, central retinal artery occlusion, branch retinalartery occlusion, carotid artery disease (CAD), frosted branch angitis,sickle cell retinopathy and other hemoglobinopathies, angioid streaks,macular oedema occurring as a result of aetiologies such as disease(e.g. diabetic macular oedema), eye injury or eye surgery; retinalischemia or degeneration produced for example by injury, trauma ortumours, uveitis, iritis, retinal vasculitis, endophthalmitis,panophthalmitis, metastatic ophthalmia, choroiditis, retinal pigmentepithelitis, conjunctivitis, cyclitis, scleritis, episcleritis, opticneuritis, retrobulbar optic neuritis, keratitis, blepharitis, exudativeretinal detachment, corneal ulcer, conjunctival ulcer, chronic nummularkeratitis, thygeson keratitis, progressive mooren's ulcer, an ocularinflammatory disease caused by bacterial or viral infection, and by anophthalmic operation, an ocular inflammatory disease caused by aphysical injury to the eye, a symptom caused by an ocular inflammatorydisease including itching, flare, oedema and ulcer, erythema, erythemaexsudativum multiforme, erythema nodosum, erythema annulare,scleroedema, dermatitis, angioneurotic oedema, laryngeal oedema, glotticoedema, subglottic laryngitis, bronchitis, rhinitis, pharyngitis,sinusitis, laryngitis or otitis media.

References herein to “back-of-eye diseases” include diseases affectingamong other the retina, macular, fovea in the posterior region of theeye. Examples of suitable “back-of-eye diseases” include but are notlimited to: macular oedema such as clinical macular oedema orangiographic cystoid macular oedema arising from various aetiologiessuch as diabetes, exudative macular degeneration and macular oedemaarising from laser treatment of the retina, age-related maculardegeneration, retinopathy of prematurity (also known as retrolentalfibroplasia), retinal ischemia and choroidal neovascularization, retinaldiseases (diabetic retinopathy, diabetic retinal oedema, retinaldetachment, senile macular degeneration due to sub-retinalneovascularization, myopic retinopathy); inflammatory diseases; uveitisassociated with neoplasms such as retinoblastoma or pseudoglioma;neovascularization following vitrectomy; vascular diseases (retinalischemia, choroidal vascular insufficiency, choroidal thrombosis,retinopathies resulting from carotid artery ischemia); andneovascularization of the optic nerve.

References herein to “front-of-eye diseases” refers to diseasesaffecting predominantly the tissues at the front-of-eye, such as thecornea, iris, ciliary body, conjunctiva etc. Examples of suitable“front-of-eye diseases” include but are not limited to: cornealneovascularization (due to inflammation, transplantation, developmentalhypoplasia of the iris, corneal diseases or opacifications with anexudative or inflammatory component, neovascularization due topenetration of the eye or contusive ocular injury; chronic uveitis;anterior uveitis; inflammatory conditions resulting from surgeries suchas LASIK, LASEK, refractive surgery, IOL implantation; irreversiblecorneal oedema as a complication of cataract surgery; oedema as a resultof insult or trauma (physical, chemical, pharmacological, etc);inflammation; conjunctivitis (e.g. persistent allergic, giant papillary,seasonal intermittent allergic, perennial allergic, toxic,conjunctivitis caused by infection by bacteria, viruses or Chlamydia);keratoconjunctivitis (vernal, atopic, sicca); iridocyclitis; iritis;scleritis; episcleritis; infectious keratitis; superficial punctuatekeratitis; keratoconus; posterior polymorphous dystrophy; Fuch'sdystrophies (corneal and endothelial); aphakic and pseudophakic bullouskeratopathy; corneal oedema; scleral disease; ocular cicatrcialpemphigoid; pars planitis; Posner Schlossman syndrome; Behcet's disease;Vogt-Koyanagi-Harada syndrome; hypersensitivity reactions; ocularsurface disorders; conjunctival oedema; toxoplasmosis chorioretinitis;inflammatory pseudotumor of the orbit; chemosis; conjunctival venouscongestion; periorbital cellulitis; acute dacryocystitis; non-specificvasculitis; sarcoidosis; and cytomegalovirus infection.

Examples of suitable “disorders associated with excessive vascularpermeability and/or edema in the eye”, e.g. in the retina or vitreous,include, but are not limited to, age-related macular degeneration (AMD),retinal edema, retinal hemorrhage, vitreous hemorrhage, macular edema(ME), diabetic macular edema (DME), proliferative diabetic retinopathy(PDR) and non-proliferative diabetic retinopathy (DR), radiationretinopathy, telangiectasis, central serous retinopathy, and retinalvein occlusions. Retinal edema is the accumulation of fluid in theintraretinal space. DME is the result of retinal microvascular changesthat occur in patients with diabetes. This compromise of theblood-retinal barrier leads to the leakage of plasma constituents intothe surrounding retina, resulting in retinal edema. Other disorders ofthe retina include retinal vein occlusions (e.g. branch or central veinocclusions), radiation retinopathy, sickle cell retinopathy, retinopathyof prematurity, Von Hippie Lindau disease, posterior uveitis, chronicretinal detachment, Irvine Gass Syndrome, Eals disease, retinitis,and/or choroiditis.

(D) Mutation of Polypeptides

The desired diversity is typically generated by varying the selectedmolecule at one or more positions. The positions to be changed areselected, such that libraries are constructed for each individualposition in the loop sequences. Where appropriate, one or more positionsmay be omitted from the selection procedure, for instance if it becomesapparent that those positions are not available for mutation withoutloss of activity.

The variation can then be achieved either by randomisation, during whichthe resident amino acid is replaced by any amino acid or analoguethereof, natural or synthetic, producing a very large number of variantsor by replacing the resident amino acid with one or more of a definedsubset of amino acids, producing a more limited number of variants.

Various methods have been reported for introducing such diversity.Methods for mutating selected positions are also well known in the artand include the use of mismatched oligonucleotides or degenerateoligonucleotides, with or without the use of PCR. For example, severalsynthetic antibody libraries have been created by targeting mutations tothe antigen binding loops. The same techniques could be used in thecontext of the present invention. For example, the H3 region of a humantetanus toxoid-binding Fab has been randomised to create a range of newbinding specificities (Barbas et al. (1992) Proc. Natl. Acad. Sci. USA,89: 4457). Random or semi-random H3 and L3 regions have been appended togermline V gene segments to produce large libraries with mutatedframework regions (Hoogenboom- & Winter (1992) R Mol. Biol., 227: 381;Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al.(1994) EMBO J, 13: 692; Griffiths et al. (1994) EMBO J, 13: 3245; DeKruif et al. (1995) J. Mol. Biol., 248: 97). Such diversification hasbeen extended to include some or all of the other antigen binding loops(Crameri et al. (1996) Nature Med., 2: 100; Riechmann et al. (1995)BiolTechnology, 13: 475; Morphosys, WO97/08320, supra).

However, since the polypeptides used in the present invention are muchsmaller than antibodies, the preferred method is to synthesise mutantpolypeptides de novo. Mutagenesis of structured polypeptides isdescribed above, in connection with library construction.

The invention is further described below with reference to the followingexamples.

EXAMPLES

Materials and Methods

Cloning of Phage Libraries

Phage libraries were generated according to Heinis et al., Nat Chem Biol2009, 5 (7), 502-7). In Heinis et al, the genes encoding a semi-randompeptide with the sequence Xaa-Cys-(Xaa)₃-Cys-(Xaa)₃- (SEQ ID No. 216),the linker Gly-Gly-Ser-Gly (SEQ ID No. 11) and the two disulfide-freedomains D1 and D2 (Kather, et al., J Mol Biol 2005, 354 (3), 666-78)were cloned in the correct orientation into the phage vector fd0D12 toobtain ‘library 3×3’. The genes encoding the peptide repertoire and thetwo gene 3 domains were step-wise created in two consecutive PCRreactions. First, the genes of D1 and D2 were PCR amplified with the twoprimer preper (5′-GGCGGTTCTGGCGCTGAAACTGTTGAAAGTAG-3′) (SEQ ID No. 12)and sfi2fo (5′-GAAGCCATGGCCCCCGAGGCCCCGGACGGAGCATTGACAGG-3′ (SEQ ID No.13); restriction site is underlined) using the vector fdg3p0ss21(Kather, et al., J Mol Biol 2005, 354 (3), 666-78) as a template.Second, the DNA encoding the random peptides was appended in a PCRreaction using the primer sficx3ba:5′-TATGCGGCCCAGCCGGCCATGGCANNKTGTNNKNNKNNKTGCNNKNNKNNKNNKTGTNNKGGGCGGTTCTGGCGCTG-3′ (SEQ ID No. 14) (restriction site is underlined),and sfi2fo. The ligation of 55 and 11 μg of SfiI-digested fd0D12 plasmidand PCR product yielded 5.6×10⁸ colonies on 10 20×20 cm chloramphenicol(30 μg/ml) 2YT plates. Colonies were scraped off the plates with 2YTmedia, supplemented with 15% glycerol and stored at −80° C. Constructionof the libraries described herein employed the same technique togenerate the semi-random peptidePro-Ala-Met-Ala-Cys-(Xaa)₃-Cys-(Xaa)₃-Cys (SEQ ID No. 15) for a 3×3library for example, and therefore replaced the sficx3ba primer sequencewith:

5′-TATGCGGCCCAGCCGGCCATGGCATGTNNKNNKNNKTGCNNKNNKNNKTGTGGCGGTTCTGGCGCTG-3′ (SEQ ID No. 16). Libraries with other loop lengths weregenerated following the same methodology.

Phage Selections

Glycerol stocks of phage libraries were diluted to OD₆₀₀=0.1 in 500 ml2YT/chloramphenicol (30 μg/ml) cultures and phage were produced at 30°C. over night (15-16 hrs). Phage were purified and chemically modifiedas described in Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7Biotinylated hPK (3 μg) (IHPKA, from human plasma, Innovative Research,Novi, Mich., USA) was incubated with 50 μl pre-washed magneticstreptavidin beads (Dynal, M-280 from Invitrogen, Paisley, UK) for 10minutes at RT. Beads were washed 3 times prior to blocking with 0.5 mlwashing buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mMCaCl₂) containing 1% BSA and 0.1% Tween 20 for 30 minutes at RT withrotation. Chemically modified phage (typically 10¹⁰-10¹¹ t.u. dissolvedin 2 ml washing buffer) were concomitantly blocked by addition of 1 mlwashing buffer containing 3% BSA and 0.3% Tween 20. Blocked beads werethen mixed with the blocked chemically modified phage and incubated for30 minutes on a rotating wheel at

RT. Beads were washed 8 times with washing buffer containing 0.1% Tween20 and twice with washing buffer before incubation with 100 μl of 50 mMglycine, pH 2.2 for 5 minutes. Eluted phage were transferred to 50 μl of1 M Tris-Cl, pH 8 for neutralization, incubated with 30 ml TG1 cells atOD₆₀₀=0.4 for 90 minutes at 37° C. and the cells were plated on large2YT/chloramphenicol plates. One or two additional rounds of panning wereperformed using the same procedures. In the second round of selection,neutravidin-coated magnetic beads were used to prevent the enrichment ofstreptavidin-specific peptides. The neutravidin beads were prepared byreacting 0.8 mg neutravidin (Pierce, Rockford, Ill., USA) with 0.5 mltosyl-activated magnetic beads (Dynal, M-280 from Invitrogen, Paisley,UK) according to the supplier's instructions.

Cloning and Expression of Human, Monkey and Rat PK

The catalytic domain of human, monkey and rat PK was expressed inmammalian cells as an inactive precursor having a pro-peptide connectedN-terminally via a proTEV cleavage site to the catalytic domain. Theexpression vector was cloned and the protein expressed, activated andpurified as described as follows. Synthetic genes coding for a PK signalsequence, a polyhistidine tag, a proTEV cleavage site, mature catalyticdomain of PK and a stop codon were purchased from Geneart (Regensburg,Germany) (Supplementary materials). Plasmid DNA containing the syntheticgenes for human, monkey (Macaca mulatta) and rat PK was prepared and thegene transferred into the pEXPR-IBA42 mammalian expression vector (IBABiotechnology, Göttingen, Germany) using the restriction enzyme pairXhoI and HindIII (Fermentas, Vilnius, Latvia) and T4 DNA ligase(Fermentas). The ligated plasmids were transformed into XL-1 blueelectrocompetent cells (Stratagene, Santa Clara, USA) and plated onto2YT agar plates containing ampicillin (10 μg/ml). DNA from the threeexpression vectors (termed mPK, rPK and hPK) was produced and thecorrect sequences confirmed by DNA sequencing (Macrogen, Seoul, SouthKorea).

The three orthologous plasma Kallikreins were expressed in mammaliancells as follows. 50 ml of suspension-adapted HEK-293 cells were grownin serum-free ExCell 293 medium (SAFC Biosciences, St. Louis, Mo.) inthe presence of 4 mM glutamine and the histone deacetylase inhibitorvalproic acid (3.75 mM) in an orbitally shaken 100 ml flask at 180 rpmin an ISF-4-W incubator (Kühner A G, Birsfelden, Switzerland) at 37° C.in the presence of 5% CO₂. The embryonic kidney (HEK-293) cells at highcell density (20×10⁶ cells/ml) (Backliwal, et al/. Biotechnol Bioeng2008, 99 (3), 721-7) were transfected with the three plasmids (300μg/ml) using linear polyethylenimine (PEI, Polysciences, Eppenheim,Germany). At the end of the 7-day production phase, cells were harvestedby centrifugation at 2′500 rpm for 15 min at 4° C. Any additional celldebris was removed from the medium by filtration through 0.45 μm PESmembranes (Filter-top 250 ml low protein binding TPP). Thepolyhistidine-tagged protein was purified by Ni-affinity chromatographyusing Ni-NTA resin, washing buffer (500 mM NaCl, 25 mM Na₂HPO₄, pH7.4)and elution buffer (500 mM NaCl, 25 mM Na₂HPO₄, pH 7.4, 500 mMimidazole). The protein was partially activated with (50 units) proTEV(Promega, Madison, Wis., USA) and additionally purified by Ni-affinitychromatography and gel filtration (PD10 column, 150 mM NaCl, 0.5 mMEDTA, 50 mM HEPES, pH 7).

Development of Polypeptides with Improved Binding Activity

Randomisation of Individual Positions

Library Construction:

In order to map the amino-acids in the Kallikrein binding bicyclicpeptides a set of small libraries was constructed. For a bicyclecomprised of 2 loops of 5 residues, 10 separate libraries were generatedeach with randomisation at a particular codon in the peptide sequence.Oligonucleotides were designed for each library in order to mutate thephage genome DNA by site-directed mutagenesis. The mutagenesisincorporated randomisation of the codon of interest (change to NNS), andremoval of a unique ApaL1 restriction site from the template genomesequence. The mutagenesis product was purified using QIAgen QIAquick PCRpurification kit with elution into ultrapure water. Each library wasused to separately transform TG1 E coli by electroporation with a BioRadMicropulser machine (Ec1 program) and 1 mm BioRad cuvette. After 1 hourrecovery at 37 C in 1 ml SOC media, the library transformants were grownovernight in 25 ml 2TY broth containing antibiotic to selectively growlibrary transformants only. The bacteria were harvested bycentrifugation and the library phage DNA was purified from the E coliusing a QIAgen Plasmid Plus Midi kit and eluted in distilled water. Thepurified DNA was digested with ApaL1 for 2 hours in New England Biolabsbuffer 4 to remove the parent material. After digestion, the DNA wasrepurified using QIAgen PCR purification kit (as above) and used totransform TG1 (electroporation; as described above). Following the 1hour recovery in SOC, transformants were plated on LB-agar platescontaining selective antibiotic and colonies allowed to grow overnightat 37 C.

Assay of Binding of Individual Clones:

Library transformant colonies were picked at random and grown asindividual cultures in 2TY broth containing selective antibiotic. Thepicked colonies were DNA-sequenced using a QIAgen PyroMark Q96 DNAsequencer to reveal the amino-acid substitution present in each clone.Where isolated, a clone of each unique substitution was assayed forhuman plasma Kallikrein binding as follows. The phage-containingsupernatant was harvested from the culture and phage were cyclised withtris bromomethyl benzene(TBMB) based on the methods of Heinis et al(Nature Chemical Biology vol. 5 pp 502-507 (2009)). The purified phagefrom this process were assayed for binding to biotinylated human plasmaKallikrein using a homogeneous plate-based binding assay; assay read-outmeasured on a BMG Labtech Pherastar FS plate reader. The quantitativebinding data from triplicate assay samples was averaged (mean) andexpressed as signal:background (where background was a sample assayedwith no target material). The signal:background was expressed as a % ofthe parallel parent sample. Error bars denote standard deviation of themean. Assays shown are representative of at least 2 independentexperiments. The assay data was correlated with the peptide sequences.Substitutions marked in grey were not tested (a clone was not isolatedfrom the random library sampling). A sample of a non-binding (arbitrary)bicycle was assayed in parallel to illustrate the assay baseline.

Randomisation of Peptide Domains

Library Construction:

Small phage libraries were generated according to the methods of Heiniset a/as described in ‘Cloning of phage libraries’ above. The sficx3baprimer was modified such that the bicycle-encoding portion was based ona parent 5×5 bicycle (5×5: two 5-residue loops) DNA sequence with only4-6 codons randomized to NNS. The randomized codons were those encodingthe peptide domain/motif of interest.

Assay of Binding of Individual Clones:

Library transformant colonies, or selection output colonies, were pickedand grown as individual cultures in 2TY broth containing selectiveantibiotic. The picked colonies were DNA-sequenced using a QIAgenPyroMark Q96 DNA sequencer to reveal the amino-acid substitution presentin each clone, and were assayed for human plasma Kallikrein binding asfollows. The phage-containing supernatant was harvested from the cultureand phage were cyclised with tris bromomethyl benzene (TBMB) based onthe methods of Heinis et al (Nature Chemical Biology vol. 5 pp 502-507(2009)). The purified phage from this process were assayed for bindingto biotinylated human plasma Kallikrein using a homogeneous plate-basedbinding assay; assay read-out measured on a BMG Labtech Pherastar FSplate reader. The quantitative binding data from duplicate assay sampleswas averaged (mean) and expressed as signal:background. Assay data shownis representative of at least 2 independent experiments. The assay datawas correlated with the peptide sequences.

Synthesis and Purification of Bicyclic Peptides

Peptide sequences are shown in Tables 4 to 6. The core peptide sequenceswere Ac-C₁ S₁W₂P₃A₄R₅ C₂ L₆H₇Q₈D₉L₁₀ C₃ —NH₂ (SEQ ID No. 7) (denoted asAc- (06-34-18)(TMB)-NH2) and Ac—C₁ S₁F₂P₃Y₄R₅ C₂ L₆H₇Q₈D₉L₁₀ C₃ —NH₂(SEQID No. 8) (denoted as Ac-(06-34-18)(TMB)-NH2 Phe2 Tyr4).

Peptide synthesis was based on Fmoc chemistry, using a Symphony peptidesynthesiser manufactured by Peptide Instruments. Standard Fmoc-aminoacids were employed (Sigma, Merck), with the following side chainprotecting groups: Arg(Pbf); Asn(Trt); Asp(OtBu); Cys(Trt); Glu(OtBu);Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc), Tyr(tBu)(Sigma). The coupling reagent was HCTU (Pepceuticals),diisopropylethylamine (DIPEA, Sigma) was employed as a base, anddeprotection was achieved with 20% piperidine in DMF (AGTC). Syntheseswere performed at 100 umole scale using 0.37 mmole/gr Fmoc-Rink amide AMresin (AGTC), Fmoc-amino acids were utilised at a four-fold excess, andbase was at a four-fold excess with respect to the amino acids. Aminoacids were dissolved at 0.2 M in DMF, HCTU at 0.4 M in DMF, and DIPEA at1.6 M in N-methylpyrrolidone (Alfa Aesar). Coupling times were generally30 minutes, and deprotection times 2×2.5 minutes. Fmoc-N-methylglycine(Fmoc-Sar-OH, Merck) was coupled for 1 hr, and deprotection and couplingtimes for the following residue were 20 min and 1 hr, respectively.After synthesis, the resin was washed with dichloromethane, and dried.Cleavage of side-chain protecting groups and from the support waseffected using 10 mL of 95:2.5:2.5:2.5 v/v/v/wTFA/H₂O/iPr3SiH/dithiothreitol for 3 hours. Following cleavage, thespent resin was removed by filtration, and the filtrate was added to 35mL of diethylether that had been cooled at −80 deg C. Peptide pellet wascentrifuged, the etheric supernatant discarded, and the peptide pelletwashed with cold ether two more times. Peptides were then resolubilisedin 5-10 mL acetonitrile-water and lyophilised. A small sample wasremoved for analysis of purity of the crude product by mass spectrometry(MALDI-TOF, Voyager DE from Applied Biosystems). Followinglyophilisation, peptide powders were taken up in 10 mL 6 M guanidiniumhydrochloride in H₂O, supplemented with 0.5 mL of 1 M dithiothreitrol,and loaded onto a C8 Luna preparative HPLC column (Phenomenex). Solvents(H₂O, acetonitrile) were acidified with 0.1% heptafluorobutyric acid.The gradient ranged from 30-70% acetonitrile in 15 minutes, at aflowrate of 15/20 mL/min, using a Gilson preparative HPLC system.Fractions containing pure linear peptide material (as identified byMALDI) were combined, and modified with trisbromomethylbenzene (TBMB,Sigma). For this, linear peptide was diluted with H₂O up to ˜35 mL, ˜500uL of 100 mM TBMB in acetonitrile was added, and the reaction wasinitiated with 5 mL of 1 M NH4HCO3 in H₂O (pH 8). The reaction wasallowed to proceed for ˜30-60 min at RT, and lyophilised once thereaction had completed (judged by MALDI). Following lyophilisation, themodified peptide was purified as above, while replacing the Luna C8 witha Gemini C18 column (Phenomenex), and changing the acid to 0.1%trifluoroacetic acid. Pure fractions containing the correct TMB-modifiedmaterial were pooled, lyophilised and kept at −20 deg C. for storage.

Non-natural amino acids were acquired from the sources set forth inTable 7.

Bulky or hindered amino acids (NMe-Ser, NMe-Trp, NorHar, 4PhenylPro,Agb, Agp, NMe-Arg, Pen, Tic, Aib, Hyp, NMe-Ala, NMe-Cys, 4,4-BPAI,3,3-DPA, Dpg, 1NAI, 2NAI, Aze, 4BenzylPro, Ind) were usually coupled for1 hours (20 min deprotection), and 6 hrs for the residue that followed(20 min deprotection). HCTU was used as a coupling reagent as before.Scale was usually at 50 umole.

Synthesis of Peptoids, Reduced Amide Bonds and α-N-Methylated Peptides

Peptoids (i.e. N-His7) were synthesised according to the schemedeveloped by Zuckermann et al. (Ronald N. Zuckermann, Janice M. Kerr,Stephen B. H. Kent, Walter H. Moos, Efficient method for the preparationof peptoids [oligo(N-substituted glycines)] by submonomer solid-phasesynthesis Journal of the American Chemical Society, (1992), 114(26),10646-10647).

Reduced amide pseudo peptide bonds were synthesised according to thescheme in Sasaki Y, Coy D H., Solid phase synthesis of peptidescontaining the CH2NH peptide bond isostere, Peptides. 1987January-February; 8(1):119-21.

N-methylated peptides (NMe-His, NMe-Gln) were synthesised using amodified form of the Mitsunobu reaction, according to a scheme describedin Nature Protocols, “Synthesis of N-methylated cyclic peptides”, byChatterjee et al, 7(3) 2012, pp 432. All other N-methylated amino acids(NMe-Leu, NMe-Asp) were obtained as Fmoc precursors from commercialsources.

Enzyme Assays

Functional enzyme assays were conducted in 10 mM Tris HCl, 150 mM NaCl,10 mM MgCl2, 1 mM CaCl2) and 1 mg/mL BSA (all Sigma UK) pH7.4 at 25° C.in solid black 96 well plates. Briefly 26.5 pM human plasma Kallikrein(purchased from Stratech, UK) or 500 pM rat plasma Kallikrein (expressedand purified in house) were incubated in the absence or presence ofincreasing concentrations of test peptide for 15 minutes before additionof the fluorogenic substrate Z-PheArg-AMC (Enzo Lifesciences UK) to afinal assay concentration of 100 μM in 4% DMSO. Release of AMC wasmeasured using a Pherastar FS (BMG Labtech), excitation 360 nm, emission460 nm. The rate of the linear phase of the reaction, typically 5 to 45minutes, was calculated in MARS data analysis software (BMG labtech).The rate was then used to calculate the IC50 and Ki in Prism (GraphPad).A four parameter inhibition non-linear regression equation was used tocalculate the IC50. The One site-fit Ki equation used to calculate theKi, constraining the Ki to the Km for the substrate which is 150 μM. AllKi/IC50 values are the mean of at least two independent experiments, andat least three for peptides with Ki values lower than 1 nM.

Peptides were dissolved as the TFA-salts in their powder form, and stocksolutions were usually prepared in water. All solutions were centrifugedand filtered (20 μm syringe filters) prior absorption measurement at 280nm. Extinction coefficients were calculated based on the Trp/Tyr contentof the peptide, and that of TMB (the TMB core, when contained in apeptide, has an ε of ˜300 M⁻¹cm⁻¹). For peptides containing non-naturalamino acids with suspected chromophoric properties (i.e. NorHar,4PhenylPro, 3Pal, 4Pal, Tic, 4GuanPhe, 4,4-BPAI, 3,3-DPA, 1NAI, 2NAI,4BenzylPro, Ind) concentrations were determined by weighing the powderand dissolving the peptide in a defined quantity of water. These wereprepared independently, twice, for peptides with a Ki to Kallikrein at 1nM or less.

Plasma Stability Profiling

Three methods were employed to assess the stability of bicycles(peptides conjugated to molecular scaffolds) in plasma.

Method #1:

A rapid plasma stability profiling assay was developed that employedmass spectrometric detection (MALDI-TOF, Voyager DE, Applied Biosystems)of the parent mass, until the time when the parent peptide mass was nolonger observable. Specifically, 200 uM of peptide was incubated in thepresence of 35% rat or human plasma (Sera labs, using citrate asanticoagulant) at 37 deg C., which was supplemented with 1×PBS (derivedfrom a 10×PBS Stock, Sigma). At various time points (i.e. t=0, 3, 24hrs, henceafter daily up to 10 days), 2 uL of sample was added to 18 uLof 30 mM ammonium bicarbonate in a 1:1 mixture of acetonitrile:H₂O.Samples were frozen at −80 deg C. until the time of analysis. For massspectrometric analysis that determines the approximate detection windowof the peptide, the acetonitrile:H₂O-diluted sample of a given timepoint was spotted directly (0.7 uL) onto the MALDI plate. Matrix(alpha-cyanocinnamic acid, Sigma, prepared as a saturated solution in1:1 acetonitrile:water containing 0.1% trifluoroacetic acid) was layeredover the sample (1 uL). At a similar laser intensity setting on theMALDI TOF, the time could then be determined until parent peptide was nolonger detectable. It should be noted that this is a qualitative assayserves to detect relative changes in plasma stability.

Method #2:

To obtain stability data more rapidly, peptides were also assessed in95% plasma. Here, PBS was omitted, and a 1 mM peptide stock (in DMSO)was directly diluted into plasma (i.e. 2.5 uL stock into 47.5 uLplasma), giving a final concentration of 50 uM. 5 uL samples were takenat appropriate time points and frozen at −80 deg C. For analysis, thesamples were defrosted, mixed with 15 uL of 1:1 acetonitrile:methanol,and centrifuged at 13 k for 5 min. 5 uL of the peptide-containingsupernatant was aspirated and mixed with 30 mM ammonium bicarbonate in a1:1 mixture of acetonitrile:H₂O. 1 uL of this was then spotted on theMALDI plate and analysed as described above. As above, it should benoted that this is a qualitative assay serves to detect relative changesin plasma stability.

Method #3:

To obtain plasma stability quantitatively, peptide stock solutions (1 mMin DMSO) were shipped to Biofocus, UK, who performed the analysis.Peptides were diluted to 100 uM with water, and diluted 1:20 in plasma(5 uM final concentration, with the plasma at 95%), sampled asappropriate, precipitated as above, and quantified using a Waters XevoTQ-MS.

Example 1: Identification of Preferred Residues for Binding Activity

From the examples of 5×5 peptides shown in Table 4 it is possible toidentify amino acids that are conserved between peptides with bindingactivity. To determine which residues were preferred for bindingactivity, representatives from two of the identified families ofpeptides were studied further. These were peptides 06-34, whichcomprises a CXWPARC (SEQ ID No. 17) motif in the first loop of thebicycle, and peptide 06-56, which comprises a CGGxxNCR (SEQ ID No. 18)motif across both loops of the bicycle. For each peptide sequence, a setof 10 phage libraries was created in which 9 of the loop residues werekept constant and the other residue was randomised so that anyamino-acid could be expressed in the library at that position. (See‘Randomisation of individual positions—Library construction’ in Methodsabove.) For each library a set of 20 randomly selected phage clones werescreened for binding to human Kallikrein in a phage binding assay toidentify the critical residues for target binding. (See ‘Randomisationof individual positions—Assay of binding of individual clones’ inMethods above.) The data from this experiment are shown in FIGS. 4-6.

For peptide 06-34 (FIG. 4), it is clear that Arg1 of the bicycle can bereplaced with a variety of different amino-acids and binding to humanplasma Kallikrein is retained or enhanced. By contrast, replacement ofresidues 2, 3, 4, 5 (Trp2, Pro3, Ala4, Arg5) by most amino acids greatlyreduced the signal seen in an assay that was set up with a stringentcut-off for high affinity binders. Val6 can be replaced by manydifferent amino-acids and binding activity is retained or enhanced.Replacement of other residues in the second loop indicated that onlyLeu10 could be replaced by a variety of different amino-acids whilstretaining activity. Positions 7, 8, and 9 have limited capacity forsubstitution and no substitutions were identified that enhanced binding.

For peptide 06-56 (FIGS. 5 and 6) it is clear that glycines at position1 and 2 are the greatly preferred residues for binding to plasmaKallikrein as are arginine, tryptophan and threonine at positions 6, 8,and 9. Glutamine at position 4 and threonine at position 10 can bereplaced by a variety of residues whilst retaining good bindingactivity. The three remaining residues—proline at position 1, asparagineat position 5 and threonine at position 7 have limited capacity forsubstitution.

Analysis of Amino-Acid Replacements

From the preceding analysis it is apparent that for 06-34, position oneand position six can be replaced by a variety of amino-acids and stillretain binding activity equal or greater than that of the parentpeptide. To evaluate whether these observations would hold with isolatedsynthetic peptides, a set of peptides was designed according to thefindings in FIG. 4, where Arg1 was replaced by a serine, and where Val6was substituted by either threonine, methionine or leucine. Peptidesemploying the various combinations of these substitutions were alsosynthesised. These substitutions produced a greater binding signal inthe assay (Table 5).

All of the variant synthetic peptides had approximately equivalent orenhanced activity against human plasma Kallikrein in enzyme inhibitionassays compared to the 06-34 parent peptide, indicating that this typeof analysis could be used to fine-tune target binding affinities, andsuggesting a route to identifying lead peptide candidates of very highpotencies.

The peptides were also tested against rat plasma Kallikrein in isolatedenzyme assays. Substitution of Arg1 to Ser1 had a marginal impact onactivity against rat Kallikrein, whereas substitutions of Val6 tothreonine, methionine or leucine generated peptides with markedlyincreased potency against rat plasma Kallikrein. Activity to humanKallikrein was fully retained. Thus, by determining positions amenableto substitutions, peptides with desirable properties, such as targetorthologue cross-reactivity, can be identified.

To demonstrate the possibility of replacing these two positions withnon-natural amino acids so as to have the capacity to introducefunctionalities or properties that are not present in the parentpeptide, Arg1 and Val6 in 06-34-03 were replaced with either alanine orN-methylglycine (sarcosine), or with N-methyl serine on position 1, andevaluated for binding. Remarkably, as shown in Table 6, positions 1/6are amenable to removal of the side chain altogether, as the R1A/V6A(06-34-03 Ala1,6) peptides retained full potency compared to the parent.Replacement of residues 1,6 with N-methylglycine (06-34-03 NMeGly1,6)caused a reduction in potency, however the binding affinity remained inthe low nanomolar range. Introduction of an N-methylserine at position 1causes a ten-fold loss in potency, but binding remains in the picomolarrange. Thus, certain positions in the bicycle can be identified thatallow changes in the peptide backbone structure or side chains, whichcould allow for deliberate enhancement of protease stability, enhancedsolubility, reduced aggregation potential, and introduction oforthologous functional groups.

Example 2: Detailed Analysis of WPAR Domain

The WPAR (SEQ ID No. 19) motif identified from Example 1 was analysed inthe context of the 06-34-03 peptide, in order to identify alternativesor improvements to the WPAR motif. A library was constructed wherepositions 1, 6, 7, 8, 9 &10 of 06-34-03 were fixed and positions 2, 3, 4& 5 were randomised (see ‘Randomisation of peptide domains—Libraryconstruction’ in Methods above). Selections against human plasmaKallikrein were performed at a variety of stringencies (see Phageselections' in Methods above). All output sequences were identified andanalysed for target binding (see ‘Randomisation of peptide domains—Assayof binding of individual clones’ in Methods above). Table 17 lists eachunique sequence, its relative abundance in the selection output(frequency), and a rank number according to target binding strength.

Table 17 shows that WPAR (SEQ ID No. 19) motif confers the best bindingto human plasma Kallikrein, although other Kallikrein binding sequencesare retrieved from selections in high abundance. These include, but arenot restricted to: WPSR (SEQ ID No. 20), WPAR (SEQ ID No. 19), WSAR (SEQID No. 21), WPFR (SEQ ID No. 22), WPYR (SEQ ID No. 23), FPFR (SEQ ID No.24), & FPFR (SEQ ID No. 24). The most effective and abundant motifs atpositions 2, 3, 4 & 5 can be summarised as: ^(W)/_(F) P×^(K)/_(R)

Table 18 shows that WPAR (SEQ ID No. 19) & WPSR (SEQ ID No. 20) weremost abundant in the more stringent selection outputs; FPFR (SEQ ID No.24) & FPYR (SEQ ID No. 25) were abundant in the lower stringencyselection outputs. This would indicate that WPAR-like sequences arestronger binders than FPFR-like sequences. Analysis of each motif(within the 06-34-03 context) in the target binding assay (FIG. 24),reveals that WPAR (SEQ ID No. 19) at positions 2, 3, 4 & 5 of the06-34-03 sequence is the optimal sequence for Kallikrein binding.

Example 3: Optimisation of the Sequence Outside the WPAR Pharmacophore

The WPAR (SEQ ID No. 19) motif and its variants have been studied withinthe context of peptide 06-34-03. FIG. 1 demonstrates that some positionsoutside of the WPAR (SEQ ID No. 19) motif can maintain Kallikreinbinding when substituted for other residues. In order to study thenon-WPAR determinants of Kallikrein binding, a phage library wasgenerated with a fixed-WPAR sequence and all other positions randomised(CxWPARCxxxxxC) (SEQ ID No. 26) as described in ‘Randomisation ofpeptide domains—Library construction’ in Methods above.

80 random library members were isolated directly from the library pool(no selection) and assayed for binding to Kallikrein at both high andlow stringency (see ‘Randomisation of peptide domains—Assay of bindingof individual clones’ in Methods above). These library members, whichcontain random sequences outside the WPAR, showed little or no bindingto human plasma Kallikrein (data not shown), indicating that thepresence of a WPAR motif alone is not sufficient to retain measureableKallikrein binding: the rest of the bicycle sequence must alsocontribute or influence the interaction.

Selections against human plasma Kallikrein were performed with thislibrary in order to study the non-WPAR determinants of Kallikreinbinding, and to isolate the optimal WPAR-containing peptide sequence.Over 150 selection output sequences were isolated and screened forbinding to human plasma Kallikrein (as described in Methods above). Thesequences were ranked in order of Kallikrein binding and the top 50sequences were aligned in Table 19. Table 19 shows that the residue atposition 1 does not affect Kallikrein binding, but a strong consensusfor Histidine is seen at position 7 (which supports findings in Example1 above). The peptide 06-34-03—derived from the work in Example 1—is oneof the best sequences. The composition of the second loop shows cleartrends which confer strong Kallikrein binding when with a WPAR motif.

The best WPAR-containing binders to human plasma Kallikrein have thetrend:

C X W P A R C ^(T)/_(L) H ^(Q)/_(T) D L C (SEQ ID No. 27)

H7, D9 and L10 are heavily conserved in WPAR-containing Kallikreinbinding sequences.

Two motifs in within the second bicycle loop (positions 6-10) wereidentified:

-   -   1. C X W P A R C T H ^(Q)/_(T) D L C (SEQ ID No. 28) (positions        6, 7 &10: “THxxL” (SEQ ID No. 219))    -   2. C X W P A R C ^(T)/_(L) H ^(Q)/_(T) D L C (SEQ ID No. 218)        (positions 7, 8, & 10: “xHxDL” (SEQ ID No. 220))

Over 120 identified human plasma Kallikrein binders (selection outputsequences) were grouped 2 different ways, according to their derivationfrom motifs “THxxL” (SEQ ID No. 219) or “xHxDL” (SEQ ID No. 220). Forall groups, the average Kallikrein binding assay signal for outputsequences was noted as a measure of Kallikrein binding for a given group(Table 20).

The Kallikrein binding assay data shown in Table 20 demonstrates that‘THxxL’ (SEQ ID No. 219) and ‘xHxDL’ (SEQ ID No. 220) motifs result inthe best Kallikrein binding when in a bicyclic peptide with a WPARmotif. The combination of the 2 motifs, ‘THxDL’ (SEQ ID No. 29), givesthe highest binding to human plasma Kallikrein and includes the ‘THQDL’(SEQ ID No. 30) second loop sequence of the 06-34-03 peptide.

Example 4: Systematic Analysis of Plasma Stability

For a Kallikrein-inhibiting bicycle, it is pertinent to obtain anadequate protease stability profile, such that it has a lowprotease-driven clearance in plasma or other relevant environments. In arapid comparative plasma stability assay (methods section, method #1)that observed the progressive disappearance of parent peptide in ratplasma, it was found that the N-terminal alanine (which is present atthe time of selections and was originally included in synthetic peptidesof lead sequences) is rapidly removed across all bicycle sequencestested by both rat and human plasma. This degradation was avoided bysynthesising a lead candidate lacking both N- and C-terminal alanines.To remove potential recognition points for amino- and carboxypeptidases,the free amino-terminus that now resides on Cys 1 of the lead candidateis capped with acetic anhydride during peptide synthesis, leading to amolecule that is N-terminally acetylated. In an equal measure, theC-terminal cysteine is synthesised as the amide so as to remove apotential recognition point for carboxypeptidasese. Thus, bicyclic leadcandidates have the following generic sequence:Ac-C₁AA₁AA₂AA_(n)C₂AA_(n+1)AA_(n+2)AA_(n+3)C₃(TMB)-NH2, where “Ac”refers to N-terminal acetylation, “—NH2” refers to C-terminal amidation,where “C₁, C₂, C₃” refers to the first, second and third cysteine in thesequence, where “AA₁” to “AA_(n)” refers to the position of the aminoacid (whose nature “AA” is defined by the selections described above),and where “(TMB)” indicates that the peptide sequence has been cyclisedwith TBMB or any other suitable reactive scaffold.

Due to the high affinity of Ac-06-34-18(TMB)-NH2 to both human (Ki=0.17nM) and rat Kallikrein (IC50=1.7 nM), we chose this Bicycle for leaddevelopment (FIG. 9, Table 1, Table 5). Using the same rapid plasmastability profiling assay described above, Ac-06-34-18(TMB)-NH2 had anobservability window of about 2 days (methods section, method #1), whichequates to a rat plasma half-life of ˜2 hrs (as determinedquantitatively by LC/MS, see below, table 23, method #3).

In an effort to identify the proteolytic recognition site(s) inAc-06-34-18(TMB)-NH2, the peptide was sampled in 35% rat plasma overtime (method #1), and each sample was analysed for the progressiveappearance of peptide fragments using MALDI-TOF mass spectrometry. Theparent mass of Ac-06-34-18(TMB)-NH2 is 1687 Da. Over time (FIG. 7, 8),fragments appear of the masses 1548.6 (M1), 1194.5 (M2), and 1107.2(M3). From the sequence of Ac-06-34-18(TMB)-NH2(Ac-C₁S₁W₂P₃A₄R₅C₂L₆H₇Q₈D₉L₁₀C₃—NH2) (SEQ ID No. 7), it can becalculated that the peak of M1 corresponds to Ac-06-34-18(TMB)-NH2lacking Arg5 (-R5). This appears to be the initial proteolytic event,which is followed by removal of the 4-amino acid segment WPAR (SEQ IDNo. 19) in Ac-06-34-18(TMB)-NH2 (M2, -WPAR), and finally the entirefirst loop of Ac-06-34-18(TMB)-NH2 is excised (M3, -SWPAR) (FIG. 8).From this data, it is evident that Arg5 of Ac-06-34-18(TMB)-NH2 is themain rat plasma protease recognition site that is responsible thedegradation of the Bicycle.

Alanine Substitutions and Scrambling of First Loop:

Having identified Arg5 in constituting the recognition site for ratplasma proteases, a campaign of chemical synthesis ofAc-06-34-18(TMB)-NH2 derivatives was undertaken with the aim ofidentifying candidates with higher plasma proteolytic stability.Crucially, such modifications should not affect the potency againsthuman or rat Kallikrein. An initial exploration regarding the role ofthe WPAR (SEQ ID No. 19) sequence/pharmacophore (FIG. 9, 10) wasperformed by replacing W₂P₃ with A₂A₃ or A₂Q₃ and by scrambling parts orthe entire first loop of the bicycle. Table 8 below shows the sequencesand the respective affinities against Kallikrein.

From these data it is clear that concomitant removal of W₂P₃dramatically reduces binding to Kallikrein by a factor of ˜100000,effectively rendering the molecule pharmacologically inert. Theimportance of the correct sequence of the amino acids is underlined bythe four scrambled peptides (Scram 1-4), as all of them display asubstantial reduction in affinity towards Kallikrein (FIG. 10).Curiously, all peptides have a roughly identical rat plasma stabilityprofile (between 1 to 2 days, method #1), indicating that plasmaprotease recognition relies on the presence of the arginine (producing adegradation pattern similar to FIG. 7), and not on its position withinthe sequence.

Next, five derivatives of Ac-06-34-18(TMB)-NH2 were generated where W₂,P₃, A₄, R₅, and C₂ were replaced with their respective D-enantiomericcounterparts (Table 9).

From the data it is clear that D-amino acid replacement of A₄, R₅, andC₂ increase peptide stability towards plasma proteases. As Arg5 excisionby rat plasma proteases appears to be the first event in peptidedegradation, the initial hydrolysis of peptide bonds will occur on theN- and/or C-terminal side of Arg5. It is plausible that replacing theamino acids to either side of Arg5 with their D-enantiomers blocksadjacent peptide bond hydrolysis through steric hindrance. Indeed, thisis an effect that has been observed previously (Tugyi et al (2005) PNAS,102(2), 413-418).

The detrimental effect of D-amino acid substitution on affinities toKallikrein is striking in all cases; losses in potencies range from 300-(D-Arg5) to 45000-fold (D-Trp2). This underlines the importance of thecorrect three-dimensional display of these sidechains to the Kallikreinbicycle binding pocket. Equally striking is the effect of D-Ala4: here,changing the orientation of a single methyl group (being the Ala sidechain) reduces the affinity 7000-fold.

N-methylations:

Next, residues in the first loop were systematically replaced with theirN-methyl counterparts. N-methylation serves as a straightforwardprotection of the peptide bond itself; however, due to the absence ofthe amide hydrogen, addition of steric bulk (the methyl group) andchanges in preferred torsional angles, losses in potencies are expected.

Table 10 summarises the data.

N-methylation of amino acids in loop 1 displays an altogether lessdrastic detrimental effect on potency. In particular, N-methylation ofArg5 still yields a single digit nanomolar binder (20-fold reduction inaffinity compared to wildtype peptide), and its rat plasma stabilityexceeds the assay time (fragmentation of the peptide in the MS was notobservable), making this an attractive improved lead candidate. As withthe D-amino acid substitutions, N-methylation of residues adjacent toArg5 imparts enhanced stability to the peptide, presumably throughsteric interference affecting protease-catalysed hydrolysis of peptidebonds N and/or C-terminal to Arg5. Of note, Ser1 can be N-methylatedwithout a significant loss in potency, indicating that the integrity ofthe peptide backbone in this position is not essential for binding.

Arginine Substitutions:

Given the importance of Arg5 in recognition by rat plasma proteases, aset of arginine analogues were tested in the Ac-06-34-18(TMB)-NH2 lead.The chemical structures are shown in FIG. 11, and the potency versusstability data is shown in Table 11.

Strikingly, all arginine analogues increase the stability of the peptidebeyond the assay window time, confirming the importance of the integrityof Arg5 in plasma protease recognition. Increasing (HomoArg) ordecreasing the length of the side chain (Agb, Agp) both decreaseaffinity, however the HomoArg analogue still yields a very good binder(Ki=2.1 nM), with enhanced stability. Lengthening the amino acidbackbone by one methylene group in Arg5 (a so-called beta-amino acid)while retaining the same side chain (β-homoArg5) also yields a binderwith enhanced stability, however at the price of a more significantreduction in affinity (Ki=8.2 nM). Replacing the aliphatic part of theArg side chain with a phenyl ring yields a resonance stabilised, bulkierand rigidified guanidyl-containing side chain (4GuanPhe). Of all the Arganalogues tested, 4GuanPhe had the greatest affinity (2-fold reductioncompared to wildtype), at an enhanced plasma stability. Interestingly,the guanidylphenyl group is structurally close to the known smallmolecule Kallikrein inhibitor benzamidine (Stürzebecher et al (1994),Novel plasma Kallikrein inhibitors of the benzamidine type. Braz J MedBiol Res. 27(8):1929-34; Tang et al (2005), Expression, crystallization,and three-dimensional structure of the catalytic domain of human plasmaKallikrein. J. Biol. Chem. 280: 41077-89). Furthermore, derivatisedPhenylguanidines have been employed as selective inhibitors of anotherserine protease, uPA (Sperl et al, (4-aminomethyl)phenylguanidinederivatives as nonpeptidic highly selective inhibitors of humanurokinase (2000) Proc Natl Acad Sci USA. 97(10):5113-8.). Thus,Ac-06-34-18(TMB)-NH2 containing 4GuanPhe5 can be viewed as a smallmolecule inhibitor, whose selectivity is imparted by the surroundingBicyclic peptide. This can comprise a principle for other bicycle-basedinhibitors, where a known small molecule inhibitor of low selectivity is“grafted” onto a Bicycle in the correct position, leading to a moleculeof superior potency and selectivity.

Modification of the Arg guanidyl group itself, either by methylation(SDMA, NDMA), removal of the positive charge (Cit, where the guanidylgroup is replaced by the isosteric but uncharged urea group) or deletionof the Arg altogether (Δ Arg) has strongly detrimental effects onKallikrein binding potency. Thus, the integrity and presence of theguanidyl group is crucial, while the nature of the sidechain connectingto the guanidyl group or backbone at Arg5 is not. Of note, Arg5 may alsobe replaced by lysine, however again at reduced affinities (see WPAK(SEQ ID No. 221) peptide).

In summary, data this far indicates that Ac-06-34-18(TMB)-NH2 employingeither HomoArg, NMeArg or 4GuanPhe as arginine replacements couldconstitute plasma stability enhanced candidates with high affinities.

Example 5: Improving the Potency of a Lead Candidate Through Non-NaturalModifications and Combination with Plasma-Stability EnhancingModifications

Improving the potency of a given bicyclic candidate can be feasiblyachieved through several mechanisms. These have been partially addressedin Example 4, and can be rewritten as follows:

-   -   1. Incorporating hydrophobic moieties that exploit the        hydrophobic effect and lead to lower off rates, such that higher        affinities are achieved.    -   2. Incorporating charged groups that exploit long-range ionic        interactions, leading to faster on rates and to higher        affinities (see for example Schreiber et al, Rapid,        electrostatically assisted association of proteins (1996),        Nature Struct. Biol. 3, 427-31)    -   3. Incorporating additional constraint into the peptide, by i.e.        -   Constraining side chains of amino acids correctly such that            loss in entropy is minimal upon target binding        -   Constraining the torsional angles of the backbone such that            loss in entropy is minimal upon target binding        -   Introducing additional cyclisations in the molecule for            identical reasons.            (for reviews see Gentilucci et al, Curr. Pharmaceutical            Design, (2010), 16, 3185-203, and Nestor et al, Curr.            Medicinal Chem (2009), 16, 4399-418).            Tryptophan and Hydrophobic Analogue Substitutions:

Initially, a range of hydrophobic amino acids were substituted into theTrp2 site to identify candidates that could replace the oxidationsensitive tryptophan, and to identify candidates that could increasepotencies (addressing the first point above). The side chains of theseamino acids are shown in FIG. 12, and affinity data is summarised inTable 12 below.

As expected, none of the modifications increase plasma stability.2-Naphtylalanine is most closely related to Trp2 and displays a potencyslightly weaker than wildtype, making this a good, oxidation-resistantreplacement for Trp2. Interestingly, 3,3-DPA2 has a structure that isvery dissimilar to Trp, yet the corresponding peptide retains highpotency. This may indicate that the Trp contacting pocket on Kallikreincould be exploited for higher affinity binding by identifying acorrectly designed hydrophobic entity.

Proline Analogues:

Next, we were interested in determining the role of Pro3 in the WPARpharmacophore in Ac-06-34-18(TMB)-NH2. 4-hydroxy- or 4-fluoro-trans(L)-proline (HyP3, 4FluoPro3) were chosen for their known property ininducing additional rigidity and helicity on the peptide backbone (FIG.13, Table 13). Additionally, the presence of the hydroxyl on HyP probesthe solvent accessibility of the proline side chain. Ki's of therespective derivatives were almost identical to that of wildtype,indicating that any effects on the peptide backbone are negligible, butalso demonstrating that the side chain is accessible. To elaborate thisfurther, two additional derivatives of Ac-06-34-18(TMB)-NH2 were tested,which contained bulky extension on the γ-carbon of the Pro3 sidechain(4Phenyl-Pro, 4Benzyl-Pro). The former displayed a striking preservationof potency, while the latter was severely impacted, demonstrating thatthe Pro side chain is accessible, but limited to distinct modificationsonly. Despite the steric bulk in these modifications, plasma stabilitywas identical to that of wildtype. Thus, these modifications do notimprove selectivity against other proteases.

To probe the effect of proline ring size on binding, the highlyconstrained 4-membered Pro analogue azetidine carboxylic acid (Aze), andthe more flexible 6-membered ring (pipecolic acid, Pip) were substitutedfor Pro3. Ac-06-34-18(TMB)-NH2 Aze3 binds Kallikrein with the highestaffinity of all derivatives so far, surpassing that of wildtype by afactor of 3 (FIG. 14, Table 13). There appears to be an inverserelationship between ring size and Ki, which would suggest thatconformational constraint at position 3 of Ac-06-34-18(TMB)-NH2 is keyto a tightly binding molecule.

The flexibility of the proline side chain in tolerating large bulkygroups is underlined by the bi/tricyclic proline analogues Tic, NorHarand Ind (FIG. 13). Particularly for the latter two cases, affinities arestill well in the one digit nanomolar range.

Finally, we sought to probe the requirement for the ring structure atPro3 altogether. To this end, we chose aminoisobutyric acid (Aib, FIG.15, Table 13), which, due to its double methyl substitution at the alphacarbon, has a strong structural effect on the neighbouring amino acidsin inducing α or 3₁₀ helicity (Toniolo et al (1993), Biopolymers 33,1061-72; Karle et a/(1990), Biochemistry 29, 6747-56). Remarkably, thisnon-natural non-cyclic amino acid is well tolerated in place of Pro3, ata Ki of 1.2 nM. Thus, the role of Pro3 in the WPAR pharmacophore is tointroduce a constraint onto the peptide backbone. This constraint can beenhanced by employing a proline analogue with reduced ring size (seeAze3). Conversely, the proline ring can be replaced relativelyefficiently with non-cyclic but structure-inducing amino acids, such asAib.

Miscellaneous Analogues:

In table 10, it was shown that Ser1 in loop 1 of Ac-06-34-18(TMB)-NH2could be N-methylated with very minor impact on potency (0.5 versus 0.17nM Ki in WT). We sought to determine whether this location tolerated alarge double substitution on Cα at position 1. To this end, Ser1 wasreplaced with Dpg (dipropylglycine) (FIG. 15). The affinity of thispeptide to Kallikrein is at 1.1 nM, indicating that position 1 is veryflexible in accommodating virtually any bulky residue. Thus, thisposition in loop 1 could be exploited for deliberate inclusion ofdesirable chemical functionalities or groups, including solubilisingamino acids, radio labels, dye labels, linkers, conjugation sites etcetera.

Several alanine analogues were also tested at position 4. As alreadyseen with the N-methyl and D-alanines (Table 10, 9), Ala4 is highlysensitive to the steric orientation at Cα, or to modification on thebackbone itself. Two more derivatives of this class underline this, aselongation of the peptide backbone at Ala4 (β-Ala4) dramatically reducesaffinity (˜20 μM). As expected from D-Ala4, Aib4 reduces affinity toalmost the same extent (289 nM, FIG. 15 and Table 14). Remarkably,extension of the Ala sidechain by one methylene (Aba4) appears toenhance the affinity to Kallikrein.

Finally, the central cysteine (Cys2) was replaced with a bulkier andmore constrained analogue, penicillamine (Pen, FIG. 15) in the hope ofincreasing proteolytic stability due to reduced spatial access to theneighbouring Arg5 protease recognition point. Indeed, rat plasmastability was slightly enhanced, however potency dropped significantly,underlining the importance of the full integrity of this structuralscaffold-connecting residue.

Combination of Plasma Stability Enhancing and Potency EnhancingNon-Natural Amino Acids into a Single Bicycle Lead

Non-natural substitutions in Ac-06-34-18(TMB)-NH2 that retainedappreciable potency and maximal rat plasma stability (as determined bymethod #1) were the Arg5 variants homoarginine (HomoArg5),4-guanidylphenylalanine (4GuanPhe5) and N-methyl arginine (NMeArg5).Non-natural substitutions in Ac-06-34-18(TMB)-NH2 that increased potencycompared to the wildtype peptide was the Pro3 analogue azetidinecarboxylic acid (Aze3) and the Ala4 analogue 2-aminobutyric acid (Aba4).Thus, Aze3, Aba4 were combined with the protease stability enhancingHomoArg5, 4GuanPhe5 and NMeArg5 to determine whether this would yieldpeptide candidates with high plasma stability and increased potency.

Table 15 and 16 present the affinities of the various constructs,together with the plasma stabilities.

Firstly, quantitative determination of rat plasma halflives (4th column,Table 15) of arginine analogue containing peptides revealed that Arg5N-methylation was most potent in protecting the peptide (t_(1/2)>20 hrs)followed by HomoArg5 and GuanPhe5. The strong protective effect of ArgN-methylation is perhaps not surprising as it directly preventshydrolysis of the peptide bond. Upon inclusion of Aze3 in thesecompounds, the affinity of these peptides could be enhanced in allcases, making Ac-(06-34-18) Aze3 HomoArg5 and Ac-(06-34-18) Aze3 NMeArg5attractive candidates for further development (Table 15, FIG. 16).

The affinity enhancing effect of Aba4 could not be reproduced in thecontext of Aze3 and any of the arginine analogues, as Ki values werehigher than those observed without Aba4. Thus, the potency enhancingeffects of Aze3 are independent of the type of Arginine substitution,while those of Aba4 are likely not.

Finally, the activity towards rat Kallikrein of these peptides isreduced significantly (Table 15). However, these values are relative andnot quantitative at this stage as the protein preparation of ratKallikrein is not trivial and contained impurities.

Example 6: Plasma Stability Enhancement of the Trp-Free FPYR KallikreinBicycle Lead and Affinity Enhancement by Aze3

From the selection output in Examples 1-4 we discovered severalsequences resembling Ac-06-34-18(TMB)-NH2 that had a high abundance, butcontained altered WPAR motifs. These were WPSR (SEQ ID No. 20) and FPYR(SEQ ID No. 25). The latter in particular is interesting as it lacks theoxidation-sensitive tryptophan.

Bicycles containing WPSR (SEQ ID No. 20), FPYR (SEQ ID No. 25), WPYR(SEQ ID No. 26) and FPAR (SEQ ID No. 31) were synthesised and comparedagainst the WPAR (SEQ ID No. 19) parent peptide (Table 21),

As expected, none of the peptides displayed a significantly differentplasma stability. The replacement of Trp2 with Phe2 incurs a 40-foldreduction in Ki, underlining the requirement of the bulkier Trp2 sidechain. However, this reduction can be compensated by replacing Ala4 withTyr4 (giving the FPYR (SEQ ID No. 25) motif), so that the affinityincreases again to almost that of the wildtype WPAR (SEQ ID No. 19)sequence (Ki=0.46 nM). Thus, there is a cooperative interplay betweenthe residues at position 2 and position 4 of the Ac-06-34-18(TMB)-NH2bicycle. Given the high target binding affinity and lack of Trp2 inAc-06-34-18(TMB)-NH2 Phe2Tyr4, this candidate was investigated forincreasing rat plasma half life employing the approach as described inthe example above. Further, we investigated the interplay between Phe2and Tyr4 by substituting these residues with non-natural amino acidanalogues.

Non-Natural Substitutions of Phe2/Tyr4 in Ac-06-34-18(TMB)-NH2 Phe2Tyr4

We performed a non-exhaustive set of syntheses incorporatingreplacements on Phe2 or Tyr4 in the Ac-06-34-18(TMB)-NH2 Phe2Tyr4 lead.Non-natural amino acids were chosen from the same set as in FIG. 12, andaffinity data is summarised in Table 22.

Here, substitution with any of the amino acids tested is generally welltolerated, regardless whether the sidechain is a heteroaromatic (3Pal,4Pal), aromatic and bulky (1Nal, 2Nal, 4,4-BPal) or a cycloaliphatic(Cha) entity. 3Pal is well tolerated at position 2 (Ki=0.91), which isinteresting as Pal contains an ionisable group (which could i.e. beexploited for formulation). It appears, however, that the originalPhe2/Tyr4 combination remains most potent.

Stabilisation of Ac-06-34-18(TMB)-NH2 Phe2Tyr4 in Rat Plasma and Effectof Azetidine Carboxylic Acid 3 Substitution

Ac-06-34-18(TMB)-NH2 Phe2Tyr4 was prepared with the homo-arginine,4-guanidylphenylalanine and N-methylarginine substitutions, in absenceand presence of Aze3. HomoArg/4Guanphe are well tolerated, with Kivalues almost identical to the parent Phe2Tyr2 peptide (Table 23,), andrat plasma stability was enhanced by a factor of 13 (t_(1/2)=12.2 hrs,Table 23, FIG. 16). Moreover, IC50 values for rat Kallikrein are similarto that of parent, indicating this to be an attractive candidate for invivo studies.

Pro3 to Aze3 substitution in the FPYR (SEQ ID No. 25) context againyielded peptide candidates with enhanced affinity, indeed a peptide witha Ki less than 1 nM was generated that would likely have a greaterhalf-life than 20 hrs in rat (Ac-(06-34-18) Phe2 Aze3Tyr4 NMeArg5).

Example 7: Improving the Human Plasma Stability of the 06-34-18 BicycleLead Candidate Through Single Amino Acid Substitutions in Loop 2

Identification of His7 as a Major Plasma Protease Recognition Site

Rat and human plasma stability of the following loop 1-modified 06-34-18peptides were determined quantitatively using LC-MS (Method #3, Table2). It is clear that the chemical modifications (HArg5, 4GuanPhe5,NMe-Arg5) that impart stability to rat plasma proteases are noteffective in the proteolytic context of human plasma.

Ac-06-34-18(TMB)-NH2 Phe2 Tyr4 (referred to as wild type peptide) wassubsequently subjected to human plasma, and analysed for fragments toappear over time. This was compared to the degradation profile observedin human plasma using the NMe-Arg5 derivative of 06-34-18 (FIGS. 18a and18b ). Fragmentation was determined according to Method #2.

According to the mass spectrometric information, endo-proteolytichydrolysis occurs on either the N- and/or C-terminal side of His7 in the06-34-18 sequence, subsequently excising Leu6 (through anexo-proteolytic activity) or His7-Gln8 (through an exo-proteolyticactivity) or all three residues together. From the nature of thefragments observed, it is likely that the initial endoproteolytichydrolysis event occurs on the peptide bond between Leu6 and His7,rather than between His7 and Gln8. Moreover, the same fragmentationpattern is observed if the rat plasma protease recognition point on Arg5is blocked with N-methylation (FIG. 18b ), indicating that theproteolytic specificity of human plasma is distinct from that of rat.

Systematic Assessment of the Role of Loop 2 Residues, and StabilisationAgainst Plasma Proteases.

Subsequently, the role of each of the loop 2 residues in terms of theireffects on human plasma stability and human kallikrein potency wasassessed.

Using experimental approaches partially outlined in the background tothe invention section, a full loop 2 amino acid scan was performed by

-   -   1) replacing each of the loop 2 amino acids with Alanine to        remove the side chain that serves as a recognition point for the        protease(s)    -   2) replacing each of the loop 2 amino acids with their D-amino        acid enantiomeric counterparts to sterically oppose access by        the degradative protease(s)    -   3) replacing some of the loop 2 amino acids with D-alanine, to        sterically oppose access to the peptide bonds by the protease(s)

The potency data for each variant are summarised in Tables 3a, 3b, and3c, respectively.

1) Alanine Scan of Loop 2 Residues

Table 3a shows that modification of His7 with Ala7 appears to reduceproteolytic degradation in the second loop, as -L, -HQ, -LHQ fragmentswere not detectable. However, proteolytic activity in loop 1 on Arg5(which was previously observed only with rat plasma) now becomesdetectable. Thus it is clear that human plasma proteases are capable ofattacking both proteolytic recognition points (Arg5 and His7). Eventhough Ala7 appears to enhance stability compared to His7 (as inwildtype peptide), its potency is reduced significantly (56-fold),bringing the Kd to 23 nM, which is not sufficiently potent for atherapeutic molecule. The remaining Ala replacements all reducepotencies to varying degrees, without increasing plasma stabilityhowever (as judged by the appearance of Loop2 degraded Bicycle Peptidefragments, Table 3a fourth column). Together, this underlines thefinding that the sidechain of His7 is the protease recognition point.

2) D-Amino Acid Scan of Loop 2 Residues

Table 3b shows the effect of replacing each of the amino acids withtheir D-enantiomeric counterparts. This modification leaves thesidechain intact, but projects it in the alternate configuration on Cα.The D-amino acids at any position within loop 2 appear to stabilise loop2 of the peptide from proteolytic degradation; however, as seen with theAla7 variant above, specificity switches now to the labile site in loop1 (Arg5). The switch of hydrolysis to Arg5 made it difficult to quantifythe effect of D-amino acid-induced stabilisation in loop2, as thisindependent process impacts the quantity of remaining parent material(addressed further below). The distal protective effect of D-amino acidsubstitution has been recognised in the literature and likely functionsby sterically reducing or preventing access to the hydrolysable bond(Tugyi et al (2005) PNAS, 102(2), 413-418). However, D-amino acidsprogressively further away from the putative His7 protease recognitionsite (i.e. D-Asp9, D-Leu10) also show a smaller quantity of fragmentscorresponding to an excised Leucine 6, which is adjacent to His7.

Notably, most of the D-amino acid variants display strongly reducedpotencies (between 130 to 9000-fold reduction in potency), underliningthe 3-dimensional and steric nature of the interaction of peptide withthe kallikrein protein. D-Asp9 appears to be the exception, in that anincrease in stability is observed, while 4 nM (9-fold reduction comparedto WT) potency is maintained. This modification could potentially be auseful stabilising substitution in a candidate therapeutic molecule.

3) D-Alanine Scan of Loop 2 Residues

Table 3c shows the effect of replacing a few select loop 2 amino acidswith D-Alanines. Removing the side chains and introducing the stericblock on Cα due to the presently introduced methyl group has stronglydetrimental effects on potencies. Nonetheless, in two cases lownanomolar affinities are retained (Ac-(06-34-18) D-Ala9, andAc-(06-34-18) Phe2 Tyr4 D-Ala9). As with D-Asp9, these modificationscould potentially be a useful stabilising substitution in a candidatetherapeutic molecule.

Replacement of Amino Acid Sidechains on Leu6, His7 and Gln8 with AminoAcid Mimetics

Subsequently a campaign of amino acid substitution of Leu6, His7 andGln8 was undertaken to evaluate the steric, charge, and structuralrequirements of these residues, and to evaluate their effect on reducingrecognition of the His7 site by plasma proteases (a structure of Loop2is presented in FIG. 19a ).

General approaches that are suitable were discussed previously in theintroductory section.

Substitutions at Position 6 in Loop2 of 06-34-18

At residue Leu6, it was sought to introduce steric bulk at Cβ of theamino acid, with the aim of reducing protease access to the scissilebond between Leu6 and His7. The structures tested are shown in FIG. 19b. The effect on potency is summarised in Table 24a. Some of themodifications show enhancement of affinities compared to the wildtypepeptide, particularly in the context of Ac-(06-34-18) (less so in thecontext of Ac-(06-34-18) Phe2 Tyr4). Specifically, these are thenon-natural amino acids phenylglycine (Phg) and cyclohexylglycine (Chg).All of these modifications appeared to somewhat reduce the proteolyticcleavage in the second loop, but the most potent stabiliser proved to betert-butylglycine (tBuGly). This peptide, Ac-(06-34-18) Phe2 Tyr4tBuGly6, showed only removal of Arg5 in the first loop when subjected tohuman plasma. Together, any of the Chg, Phg, and tBuGly modificationscould serve to enhance second loop plasma stability. In addition, weidentified two modifications (Chg, Phg) that enhance the potency towardsKallikrein.

Substitutions at Position 7 in Loop2 of 06-34-18

At residue 7, the effect on plasma stability and potency by replacingthe histidine side chain was studied employing the following mimics: Thepyridylalanines 2Pal, 3Pal, 4Pal are basic ionisable heteroaromaticmimics of the imidazole sidechain. Thienylalanine (Thi), furylalanine(FuAla), ThiAz, 1,2,4 triazolalanine, (1,2,4 Triaz), thiazolylalanine(ThiAz) are 5-membered heteroaromatic uncharged mimics of the imidazoleside chain. His1Me/His3Me are N-methyl substituted derivatives of theimidazole side chain. Dap, Agb and Agp represent positive chargereplacements of the imidazole side chain, lacking an aromatic sidechainhowever (FIG. 19c ). Table 24b summarises the potency data. Strikingly,all peptides display an enhanced stability towards plasma proteases inloop2, visible degradation occurred only in Loop 1 on Arg5. Clearly, apositive charge at position 7 is not sufficient to provide a recognitionpoint for plasma proteases (Dap, Agp, Agb). Conversely, removal of thepositive charge while retaining close mimicry of the original imidazoleside chain also removes the proteolytic recognition site. Despite thevery close mimicry of some of structures to the original imidazole ring,all of the peptide derivatives display a significantly reduced affinityto plasma kallikrein. This indicates that the intact structure of His 7is required for binding to kallikrein with full potency. Interestingcandidates that retained low-nanomolar binding were thiazolylalanine,furylalanine, and His1Me. Interesting putative candidates could behomohistidine, and imidazolylglycine.

Substitutions at Position 8 in Loop2 of 06-34-18

Residue 8 (Gln8) is more distal to the cleavage site that residesbetween Leu6 and His7. A few substitutions were tested (FIG. 19d andTable 24c). Negatively charged amino acids were introduced at thisposition so as to encourage a salt bridge to the neighbouring His7,which could reduce recognition of His7 by plasma proteases. Anadditional Loop2 sequence was tested that was present in the phageselection outputs, which also lacked a histidine 7. This loop 2 sequencewas LTTEL (SEQ ID No. 32) (referred to as Ac-(06-34-18) Phe2 Tyr4 Thr7Thr8 Glu9). Additionally, a beta amino acid was tested at position 8(bHGln8, FIG. 19d ). A positive charge amino acid was also tested atthis position so as to potentially thwart proteolytic recognition ofHis7 and subsequent degradation. While potencies of these molecules wereoften <10 nanomolar, the effect on plasma stabilisation was minor. Thesemolecules may warrant further quantitative study to determine theirprecise effects on plasma stability.

Example 8: Improving the Human Plasma Stability of the 06-34-18 BicycleLead Candidate Through Amino Acid Backbone Modifications in Loop 2

Amino acid backbone modification presents a widely recognised means bywhich the peptide backbone can be protected from protease hydrolysis(for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010),16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16,4399-418).

A well-established method in the art is to N-alkylate, for exampleN-methylate the Nα of amino acids. Table 25a shows the effect ofsystematic replacement of loop two amide backbones with theirNα-methylated counterparts. In principle, this is a very effectivehydrolysis-protective modification, as not only the peptide bond itselfis protected from proteolytic hydrolysis, but also as the bulk of themethyl group on the Nα has protective effects on neighbouring peptidebonds. Indeed, strongly protective effects on loop2 proteolysis areobservable, however potencies invariably decrease by a factor of 100 orgreater. As with D-amino acid replacement, N-alkylation protects thescissile site at His7 even when it is present at Asp9 or Leu10.

Another means by which the peptide backbone can be modified is byN-alkylation of the Nα with an appropriate amino acid side chain (termedpeptoid). The homohistidine sidechain was introduced at the Nα of His7,while concomitantly removing the side chain on Cα of His7, yielding anN-alkyl glycine. The structure of this derivative is shown in FIG. 20a(termed N-His7), within the context of the entire second loop of06-34-18.

In an attempt to modify the structure of the peptide backbone itself, areduced form of the peptide bond was introduced between Leu6 and His7(FIG. 20b ). Here, Leu6 was replaced by Ala6, and the carbonyl on thisresidue was reduced to methylene, yielding a linkage that lacks thenative peptide bond altogether. This derivative is referred to as“reduced amide” or “pseudo peptide bond”, and termed ψCH₂NH. Due to theabsence of the carbonyl on this position, the peptide cannot behydrolysed at this position and should infer favourable proteasestability characteristics to the peptide.

In the plasma assay, both peptide derivatives (Ac-(06-34-18) Phe2 Tyr4N-His and Ac-(06-34-18) Phe2 Tyr4 Ala(ψCH₂NH)6) displayed significantlyenhanced stability in loop2. Fragments observed solely corresponded todegradation in Loop 1, on Arg5. Potency data is summarised in Table 25b.As expected, the N-His derivative showed strongly reduced binding.However, the pseudo amide peptide Ac-(06-34-18) Phe2 Tyr4 Ala(ψCH2NH)6)retained a very good potency at a Kd of 1.5 nM.

Example 9: Generation of Potent, Rat and Human Plasma-Stable,Cross-Reactive Bicyclic Peptide Leads by Selective Chemical Modificationin Both Loop 1 and Loop 2

From the preceding two examples, several loop 2 modifications wereidentified that enhanced protease stability in loop2, while displaying agood potency. Retention of an appropriate potency towards rat plasmakallikrein is desirable for establishing PK/PD in animal models. Thecomparative rat potency is summarised for suitable loop2stability-enhanced candidates in Table 26a. Most of the moleculesdisplay potencies <100 nM towards rat plasma kallikrein.

In Examples 1-6, methods have been disclosed by which Loop 1 of 06-34-18can be stabilised against plasma proteases. These advantageousmodifications are combined with the plasma stability enhancingmodifications in Loop 2 (Examples 7, 8). Several representativecandidates were prepared and assessed for human/rat plasma stability, aswell as potency towards human/rat plasma kallikrein (Table 26b).

Notably, most of the peptides display, when in combination with the loop1 modifiers, better potencies towards human and rat kallikrein thanpeptides with the loop 2 modifiers alone (Table 26a). Most of thepotencies are in a range acceptable for therapeutic and PK/PD purposes(see human and rat kallikrein data, Table 26b).

Due to the introduction of HArg5 in place of Arg5 in loop1, and due tothe introduction of modifications in loop2 that reduced proteolysis atthe His 7 site, the peptides shown in Table 26a and 26b should displayenhanced stability in both human and rat plasma.

Plasma stability was assessed comparatively as before using the MALDITOF assay (Methods #1 and #2), and formation of peptidic hydrolysedfragments was observed over time (for wildtype stability, see FIGS. 18a,21a, 21b ). Compared to wildtype peptide, the multivariant peptidesAc-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 (human plasma stability:FIG. 21d , rat plasma stability: FIG. 21e ) and Ac-(06-34-18) Phe2 Aze3Tyr4 HArg5 D-Asp9 (human plasma stability: FIG. 21c ) display aremarkably enhanced stability in both human rat plasma.

Together, this shows that suitable modifications both in Loop 1 and Loop2 of 06-34-18 can be combined to yield highly potent, cross-reactive,and plasma stable bicyclic peptides, which display a profile suitablefor therapeutic purposes.

Example 10: Advantageous In Vivo Pharmacokinetic Properties of Two LeadPeptides, Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 andAc-(06-34-18) Phe2 Aze3 Tyr4 HArg5 D-Asp9

Peptides Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 (which containsthe affinity enhancing modifications Aze3 and the plasma proteasestability enhancing modifications HArg5 and the plasma proteasestability enhancing reduced amide bond between Ala6 and His 7 in Loop2)and Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 D-Asp9 (which contains theaffinity enhancing modification Aze3 and the plasma protease stabilityenhancing modifications HArg5 and D-Asp9 in Loop2) were selected forpharmacokinetic assessment in rat. The full chemical structure ofAc-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 is shown in FIG. 22.

The clearance rates from the rat circulation for both peptides wereassessed and compared to a control peptide that was composed of theAc-(06-34-18)(TMB) sequence whose amino acids were replaced by each ofthe respective D-enantiomers (the sequence thus being“(Ac-cswparclhqdlc-NH2(TMB)). This control peptide has identical atomicand sequence composition, retains similar physico-chemical properties,lacks any potency towards the kallikrein target, and importantly iscompletely resistant to proteolytic activities. Thus, the clearance fromthe rat circulation of the all D Ac-06-34-18 control peptide is drivenmostly renally, as the proteolytically-driven clearance is absent. Anyoptimised protease-stabilised and potent kallikrein-inhibiting Bicyclethat is based on the 06-34-18 sequence should therefore clear from therat circulation at a rate similar to the control All-D 06-34-18 peptide.

Peptides were applied as intravenous boli in PBS-buffered solution at1.02 mg/mL (containing 2.9% DMSO) at 5.2 mg/kg to Sprague Dawley rats,and serial blood samples were taken from each animal via temporaryindwelling tall vein cannulae at 5, 10, 20, 30, 60, 120, and 240 minutespost dose, and transferred to EDTA tubes for plasma generation. Data wasacquired for peptide “all D Ac-06-34-18” from 3 rats, for “Ac-(06-34-18)Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6” from two rats, and for “Ac-(06-34-18)Phe2 Aze3 Tyr4 HArg5 D-Asp9” from two rats.

Plasma samples were then treated with 3 volumes of a 1:1 mixture ofacetonitrile and methanol, precipitated proteins were removed bycentrifugation, and the supernatant was analysed by UPLC-MS/MS.

Quantification for peptide content in the samples was by reference to acalibration line prepared in control rat plasma. Pharmacokineticparameters were determined by non-compartmental analysis using thesoftware package PK Solutions 2.0 from Summit Research Services.

The peptide (Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 displayedan elimination half-life of 48 minutes, while the control All D 06-34-18peptide cleared at a 54 minute half-life. The volumes of distributionfor the two peptides were comparable (Table 27, FIG. 23). Thus, thepharmacologically active (Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5Ala(ψCH2NH)6 peptide is as stable as the pharmacologically inertprotease stable All D 06-34-18 control peptide, indicating that(Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 has favorableproperties for further development as a therapeutic (FIG. 23). PeptideAc-(06-34-18) Phe2 Aze3 Tyr4 HArg5 D-Asp9 cleared more rapidly, however,with an elimination half-life of ˜27 minutes.

The intravenous pharmacokinetic parameters for the 3 peptides aresummarised in Table 27.

The results underline the enhanced proteolytic stability of peptideAc-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Ala(ψCH2NH)6 in the in vivocirculation of the rat. The clearance at 5.8 mL/min/kg is very close tothe published renal filtration rate in rat (˜8-9 mL/min/kg), indicatingthat plasma protease-driven clearance of this Bicyclic peptide isvirtually absent [Jobin J, Bonjour J P, (1985) Am J Physiol.; 248(5 Pt2):F734-8].

Unless otherwise stated, any methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention. Methods, devices, and materials suitable for suchuses are described above. All publications cited herein are incorporatedherein by reference in their entirety for the purpose of describing anddisclosing the methodologies, reagents, and tools reported in thepublications that might be used in connection with the invention.

TABLE 1 Potencies, Rat plasma stability and species cross reactivity ofAc-06-34-18 as disclosed in PCT/EP2012/069898. Ki (nM) Observable in(human rat plasma, t_(1/2) (hrs) in Ki (nM) Peptide kallikrein) fordays¹ rat plasma² (rat kallikrein) Ac-(06-34-18) wildtype 0.17 2.0 2.31.8 Ac-(06-34-18) HomoArg5 2.1 >10 10.7 101 Ac-(06-34-18) 4GuanPhe50.34 >10 2.8 3.0 Ac-(06-34-18) NMeArg5 3.5 >10 >20 119.7 Ac-(06-34-18)Aze3 HomoArg5 0.14 >10 nd 14.8 Ac-(06-34-18) Aze3 4GuanPhe5 0.17 >10 ndnd Ac-(06-34-18) Aze3 NMeArg5 1.3 >10 nd 70.2 Ac-(06-34-18) Phe2 Tyr4wildtype 0.46 2 0.9 7.6 Ac-(06-34-18) Phe2 Tyr4 HomoArg5 0.77 >10 12.216.2 Ac-(06-34-18) Phe2 Tyr4 4GuanPhe5 0.40 >10 5.0 0.6 Ac-(06-34-18)Phe2 Tyr4 NMeArg5 3.56 >10 >20 64.3 Ac-(06-34-18) Phe2 Aze3 Tyr4HomoArg5 0.12 nd nd 3.0 Ac-(06-34-18) Phe2 Aze3Tyr4 4GuanPhe5 0.36 nd nd1.2 Ac-(06-34-18) Phe2 Aze3Tyr4 NMeArg5 0.97 nd nd 31.4 ¹Determinedaccording to Method #1. ²Determined according to Method #3. Tablereproduced from PCT/EP2012/069898.

TABLE 2 Summary of the ex vivo rat and human plasma stabilities ofdifferent Loop1-modified 06-34-18 peptide derivatives, as determined byMethod #3. Human Ki (nM) Rat Plasma Plasma human Stability Stability Kai(t½ in hrs) (t½ in hrs) Ac-06-34-18 Phe2 Tyr4 0.42 0.9 1.4 Ac-06-34-18Phe2 Tyr4 NMeArg5 4 >20 1.7 Ac-06-34-18 Phe2 Tyr4 0.77 12.2 2.1 HomoArg5Ac-06-34-18 Phe2 Tyr4 0.4 5 2.9 GuanidylPhe5

TABLE 3a Alanine Scan in Loop2. Resultant potencies are compared to WT,and peptide fragments observed after incubation in human plasma arenoted. Alanine Scan F (Ki_(var)/ Fragments of Ac-(06-34-18) Phe2 Tyr4 Ki(nM) Ki_(WT)) (human plasma) Ac-(06-34-18) Phe2 Tyr4 0.42 1 -LHQ (wildtype) Ac-(06-34-18) Phe2 Tyr4 Ala6 1.90 5 -LHQ, -R Ac-(06-34-18) Phe2Tyr4 Ala7 23.43 56 -R Ac-(06-34-18) Phe2 Tyr4 Ala8 1.74 4 -LHQ, -RAc-(06-34-18) Phe2 Tyr4 Ala9 1.58 4 -LHQDL, -R Ac-(06-34-18) Phe2 Tyr4Ala10 13.46 32 -LHQ, -LHQDL, -R

TABLE 3b D-amino acid Scan in Loop2. Resultant potencies are compared toWT, and peptide fragments observed after incubation in human plasma arenoted. Fragments (human D-amino acid scan Ki (nM) F (Ki_(var)/Ki_(WT))plasma) Ac-(06-34-18) Phe2 Tyr4 D-Leu6 9078.33 21615 -R, -FPYRAc-(06-34-18) Phe2 Tyr4 D-His7 129.18 308 -R, -FPYR Ac-(06-34-18) Phe2Tyr4 D-Gln8 316.90 755 -R, -FPYR Ac-(06-34-18) Phe2 Tyr4 D-Asp9 3.89 9-R, -FPYR Ac-(06-34-18) Phe2 Tyr4 D-Leu10 415.67 990 -R, -FPYR

TABLE 3c D-Alanine Scan in Loop2. Resultant potencies are compared toWT. Fragments were similar to those seen with the full D-amino acidreplacements (Table 3b). Note that both Ac-(06-34-18) and Ac-(06-34-18)Phe2 Tyr4 were also assessed. D-Alanine Scan Ki (nM) F(Ki_(var)/Ki_(WT)) Ac-(06-34-18) D-Ala6 1037.00 2469 Ac-(06-34-18)D-Ala8 11.67 28 Ac-(06-34-18) D-Ala9 0.87 2 Ac-(06-34-18) Phe2 Tyr4D-Ala6 9453.00 22507 Ac-(06-34-18) Phe2 Tyr4 D-Ala8 242.60 578Ac-(06-34-18) Phe2 Tyr4 D-Ala9 19.87 47 Ac-(06-34-18) Phe2 Tyr4 D-Ala10572.15 1362

TABLE 4 Kalli- krein Throm- SEQ Av Ki bin Factor Sequence ID No. (nM)Ic50 XIIa 06- ACAWPARCLTVDLCA A C A W P A R C L T V D L C A 33<0.1* >10000 >10000 01 06- ACRWPARCVHQDLCA A C R W P A R C V H Q D L C A34 <0.3* >10000 >10000 34 06- ACSWPARCNHQDLCA A C S W P A R C N H Q D LC A 35 0.4 >10000 >10000 57 06- ACRWPARCLTTSLCA A C R W P A R C L T T SL C A 36 0.5 >10000 >10000 59 06-54 ACRWPARCTHQNYCA A C R W P A R C T HQ N Y C A 37 0.49 >10000 >10000 (2A2)T 06- ACTWPARCTHQNWCA A C T W P A RC T H Q N W C A 38 1.2 >10000 >10000 09 06- ACFPSHDCDGRRMCA A C F P S HD C D G R R M C A 39 1.27 >10000 >10000 143 06- ACGGPQNCRTWTTCA A C G GP Q N C R T W T T C A 40 2.1 >10000 >10000 56 06- ACNWPYRCLHTDLCA A C NW P Y R C L H T D L C A 41 3.3 >10000 >10000 157 06- ACSWPYRCLHQDYCA A CS W P Y R C L H Q D Y C A 42 5.8 >10000 >10000 61 06- ACGVPYRCTHQEMCA AC G V P Y R C T H Q E M C A 43 6.9 >10000 >10000 64 T 06-ACTWPARCTMQNWCA A C T W P A R C T M Q N W C A 44 181 >10000 A2* 06-ACADPWACLFRRPCA A C A D P W A C L F R R P C A 45 1277 >10000 >10000 63 T1E6 ACAWPARCLTTSLCG A C A W P A R C L T T S L C G 46 0.16 >10000 >100002A10 ACTYPYKCLHQNLCA A C T Y P Y K C L H Q N L C A 47 4.98 1B1ACAWPAKCLTRELCA A C A W P A K C L I R E L C A 48 8.1 1F7 ACGGYNNCRAFSYCAA C G G Y N N C R A F S Y C A 49 2.2

TABLE 5 06-34 - substitutions based on identificationof non-critical residues with natural amino acids IC50 IC50 SEQ humanrat ID Peptide Sequence PK (nM) PK (nM) No. 06-34 ACRWPARCVHQDLCA* 0.197.38 34 06-34-01 AC S WPARCVHQDLCA 0.15 6.12 50 06-34-02 ACRWPARC THQDLCA 0.16 1.09 51 06-34-03 AC S WPARC T HQDLCA 0.082 0.87 52 (01 + 02)06-34-04 ACRWPARC M HQDLCA 0.075 0.56 53 06-34-05 ACRWPARC L HQDLCA0.076 0.62 54 06-34-17 AC S WPARC M HQDLCA 0.073 0.44 55 (01 + 04)06-34-18 AC S WPARC L HQDLCA 0.070 0.56 56 (01 + 05) 06-34-19 AC S WPA

C L HQDLCA 0.19 4.67 57 (01 + 05 + R/K) *Residue numbering is from leftto right, where residues 1-5 are in loop 1,and residues 5-10 are in loop2.

TABLE 6 06-34 - substitutions based on identificationof non critical residues with N- methylated amino acids Ki human SEQ PKID Peptide Sequence (nM) No. 06-34 ACRWPARCVHQDLCA 0.128 34 06-34-03- ACA WPARC A HQDLCA 0.147 58 Ala1,6 06-34-03-N- AC N-MeG WPARC 24.8 59MeGly1,6 ( N-MeG )HQDLCA 06-34-18 AC S WPARC L HQDLCA 0.040 6006-34-18-N- AC N-MeS WPARC L HQDLCA 0.560 61 MeSer1

TABLE 7 Short Supplier name Full chemical name AGTC D-AspFmoc-D-Asp(tBu)-OH Anaspec NDM-Arg Fmoc-Nwωdimethyl-L-arginine AnaspecNMe-Ser Fmoc-Nα-methyl-O-t-butyl-L-serine Anaspec NMe-TrpFmoc-Nα-methyl-L-tryptophan Anaspec NorHarFmoc-L-1;2;3;4-tetrahydro-norharman-3-carboxylic acid Anaspec 4PhenylProFmoc-(2S;4S)-4-phenyl-pyrrolidine-2-carboxylic acid Iris Biotech AgbFmoc-L-Agb(Boc)2-OH Iris Biotech Agp Fmoc-L-Agp(Boc)2-OH Iris Biotechβ-Ala Fmoc-beta-Ala-OH Iris Biotech Cit Fmoc-Cit-OH Iris Biotech D-CysFmoc-D-Cys-OH Iris Biotech β-HArg Fmoc-L-beta-HArg(Pbf)-OH Iris BiotechNMe-Arg Fmoc-L-MeArg(Mtr)-OH Iris Biotech 3Pal Fmoc-L-3Pal-OH IrisBiotech 4Pal Fmoc-L-4Pal-OH Iris Biotech Pen Fmoc-Pen(Trt)-OH IrisBiotech D-Pro Fmoc-D-Pro-OH Iris Biotech Tic Fmoc-L-Tic-OH Iris BiotechD-Trp Fmoc-D-Trp-OH Merck Novabiochem Aib Fmoc-Aib-OH Merck NovabiochemD-Ala Fmoc-D-Ala-OH Merck Novabiochem D-Arg Fmoc-D-Arg(Pbf)-OH MerckNovabiochem 4GuanPhe Fmoc-Phe(bis-Boc-4-guanidino)-OH Merck NovabiochemD-Gln Fmoc-D-Gln(Trt)-OH Merck Novabiochem D-His Fmoc-D-His(Trt)-OHMerck Novabiochem Hyp Fmoc-Hyp(tBu)-OH Merck Novabiochem D-LeuFmoc-D-Leu-OH Merck Novabiochem NMe-Ala Fmoc-L-MeAla-OH MerckNovabiochem NMe-Cys Fmoc-N-Me-Cys(Trt)-OH Merck Novabiochem SDMAFmoc-SDMA(Boc)2-ONa Merck Novabiochem HArg Fmoc-L-HArg(Boc)2-OH PeptechCorporation 4,4-BPAl Fmoc-L-4,4′-Biphenylalanine Peptech Corporation3,3-DPA Fmoc-L-3,3-Diphenylalanine Peptech Corporation DpgFmoc-Dipropylglycine Peptech Corporation 1NAl Fmoc-L-1-NaphthylalaninePeptech Corporation 2NAl Fmoc-L-2-Naphthylalanine Peptech CorporationPip Fmoc-L-Pipecolic acid Polypeptide Group Aba Fmoc-L-2-aminobutyricacid Polypeptide Group Aze Fmoc-L-azetidine-2-carboxylic acidPolypeptide Group 4BenzylPro(2S,4R)-Fmoc-4-benzyl-pyrrolidine-2-carboxylic acid Polypeptide GroupCha Fmoc-beta-cyclohexyl-L-alanine Polypeptide Group 4FluoPro(2S,4R)-Fmoc-4-fluoro-pyrrolidine-2-carboxylic acid Polypeptide GroupInd Fmoc-L-Indoline-2-carboxylic acid AGTC D-Asp Fmoc-D-Asp(tBu)-OHMerck Chemicals Thi Fmoc-L-Thi-OH Sigma 4ThiAzFmoc-β-(4-thiazolyl)-Ala-OH Santa Cruz Biotechnology 1,2,4-TriAzFmoc-3-(1,2,4-triazol-1-yl)-Ala-OH Combi-Blocks 4FuAla7Fmoc-L-2-Furylalanine Fluorochem N-His2-(1-Trityl-1H-imidazol-4-yl)-ethylamine Iris Biotech AgbFmoc-L-Agb(Boc)2-OH Iris Biotech Agp Fmoc-L-Agp(Boc)2-OH Merck 1MeHisFmoc-1-methyl-L-histidine Merck 3MeHis Fmoc-3-methyl-L-histidine SigmabHGln Fmoc-β-Homogln(Trt)-OH Iris Biotech 3Pal Fmoc-L-3Pal-OH IrisBiotech 4Pal Fmoc-L-4Pal-OH Merck Novabiochem D-Leu Fmoc-D-Leu-OH MerckNovabiochem HArg Fmoc-L-HArg(Boc)2-OH Polypeptide Group AzeFmoc-L-azetidine-2-carboxylic acid Combi Blocks Ala-H Fmoc-L-Alaninealdehyde Combi Blocks FuAla Fmoc-L-2-furylalanine

TABLE 8 Observ- KI (nM) able (human in rat SEQ kalli- plasma, ID PeptideSequence krein) for days No. Ac-(06-34- Ac-CSWPARCLHQDLC 0.17 2 22218) wildtype Ac-(06-34- Ac-CSAAARCLHQDLC 18545 1  61 18) A2A3 Ac-(06-34-Ac-CSAQARCLHQDLC 15840 1  62 18) A2Q3 Ac-(06-34- Ac-CPSAWRCLHQDLC 1091 2 63 18) Scram1 Ac-(06-34- Ac-CWASPRCLHQDLC 11355 2  64 18) Scram2Ac-(06-34- Ac-CAPWSRCLHQDLC 1892 1  65 18) Scram3 Ac-(06-34-Ac-CWARSPCLHQDLC 67500 1  66 18) Scram4

TABLE 9 Comparative effects of D-amino acid substitution on potency andrat plasma stability. Observable Ki (nM) (human in rat plasma, Peptidekallikrein) for days Ac-(06-34-18) wildtype 0.17 2 Ac-(06-34-18) D-Trp27558 2 Ac-(06-34-18) D-Pro3 680 3 Ac-(06-34-18) D-Ala4 1203 >10Ac-(06-34-18) D-Arg5 52 >10 Ac-(06-34-18) D-Cys2 234 >10

TABLE 10 Comparative effects of N-methylation of loop 1 residues andCys2 on potency and rat plasma stability. Observable Ki (nM) (human inrat plasma, Peptide kallikrein) for days Ac-(06-34-18) wildtype 0.17 2Ac-(06-34-18) NMeSer1 0.5 3 Ac-(06-34-18) NMeSer1, NMeAla4 444 >10Ac-(06-34-18) NMeTrp2 228 5 Ac-(06-34-18) NMeAla4 343 >10 Ac-(06-34-18)NMeArg5 3.5 >10 Ac-(06-34-18) NMeCys2 418 10

TABLE 11 Comparative effects of arginine analogues inAc-06-34-18(TMB)-NH2 on potency and stability. Note that the Δ Argmodification did not display any inhibition up to 100 μM peptide. Ki(nM) Observable (human in rat plasma, Peptide kallikrein) for daysAc-(06-34-18) wildtype 0.17 2 Ac-(06-34-18) HomoArg5 2.1 >10Ac-(06-34-18) Agb5 83 >10 Ac-(06-34-18) Agp5 1770 >10 Ac-(06-34-18)βhomoArg5 8.2 >10 Ac-(06-34-18) 4GuanPhe5 0.3 >10 Ac-(06-34-18) SDMA51415 >10 Ac-(06-34-18) NDMA5 510 >10 Ac-(06-34-18) Cit5 7860 >10Ac-(06-34-18) Δ Arg5 >100000 >10

TABLE 12 Comparative affinity effects of hydrophobic amino acidssubstituting Trp2 in Ac-06-34-18 (TMB)-NH2 Observable Ki (nM) (human inrat plasma, Peptide kallikrein) for days Ac-(06-34-18) wildtype 0.17 2Ac-(06-34-18) 1NAL2 10.7 2 Ac-(06-34-18) 2NAL2 0.50 2 Ac-(06-34-18)3Pal2 59 2 Ac-(06-34-18) 4Pal2 72 2 Ac-(06-34-18) Cha2 4.7 2Ac-(06-34-18) 4,4,BPal2 464 2 Ac-(06-34-18) 3,3-DPA2 1.5 2 Ac-(06-34-18)NorHar2 24 2

TABLE 13 Comparative affinities obtained for proline derivatives withgamma-carbon substituents, analogues of varying ring sizes, bi/tricyclicderivatives, and constrained amino acids such as Aib Ki Observable (nM)(human in rat plasma, Peptide kallikrein) for days Ac-(06-34-18)wildtype 0.17 2 Ac-(06-34-18) HyP3 0.41 2 Ac-(06-34-18) 4FluoPro3 0.24 2Ac-(06-34-18) 4Phenyl Pro3 0.58 2 Ac-(06-34-18) 4Benzyl Pro3 191 2Ac-(06-34-18) Aze3 0.06 2 Ac-(06-34-18) Pip3 0.26 2 Ac-(06-34-18) Tic313.51 2 Ac-(06-34-18) NorHar3 2.99 2 Ac-(06-34-18) Ind3 1.35 2Ac-(06-34-18) Aib3 1.20 2

TABLE 14 Comparative effects of miscellaneous substitutions of Ser1,Ala4, and Cys2 Ki Observable (nM) (human in rat plasma, Peptidekallikrein) for days Ac-(06-34-18) wildtype 0.17 2 Ac-(06-34-18) Dpg11.09 2 Ac-(06-34-18) Aba4 0.07 2 Ac-(06-34-18) β-Ala4 17450 10Ac-(06-34-18) Aib4 289 7 Ac-(06-34-18) Cys2ToPen2 2162 5

TABLE 15 Comparative enhancement in potency induced by incorporation ofAze3 in plasma-stabilised candidates. Ki (nM) Observable in t_(1/2)(hrs) IC50 (nM) (human rat plasma, in rat (rat Peptide kallikrein) fordays¹ plasma² kallikrein)³ Ac-(06-34-18) 0.17 2.0 2.3 1.7 wildtypeAc-(06-34-18) 2.1 >10 10.7 64 HomoArg5 Ac-(06-34-18) 0.34 >10 2.8 214GuanPhe5 Ac-(06-34-18) 3.5 >10 >20 98 NMeArg5 Ac-(06-34-18) Aze30.14 >10 nd nd HomoArg5 Ac-(06-34-18) Aze3 0.17 >10 nd nd 4GuanPhe5Ac-(06-34-18) Aze3 1.30 >10 nd nd NMeArg5 ¹Comparative stabilitiesestimated according to method #1. ²The true half-life of peptidestabilities in rat plasma was determined according to method #3. ³IC50values are relative, not absolute.

TABLE 16 Effect on potency upon inclusion of Aba4 in peptides containingAze3 and the plasma-stabilising modifications NMeArg5, HomoArg5, and4GuanPhe5. Ki (nM) (human Peptide kallikrein) Ac-(06-34-18) wildtype0.17 Ac-(06-34-18) Aze3 Aba4 NMeArg5 2.8 Ac-(06-34-18) Aze3 Aba4HomoArg5 0.9 Ac-(06-34-18) Aze3 Aba4 4GuanPhe5 0.2

TABLE 17 42 unique Kallikrein binders were identified fromselections using a randomised WPAR motif atpositions 2, 3, 4 & 5 within the 06-34-03 sequence.The sequences were ranked according to Kallikreinbinding and the relative abundance in the totalselection outputs was noted. SEQUENCE RANK FREQUENCY SEQ ID NO.CSWPARCTHQDLC 1 24 67 CSWPSRCTHQDLC 2 51 68 CSFPFRCTHQDLC 3 17 69CSWLARCTHQDLC 4 8 70 CSFPYRCTHQDLC 5 12 71 CSFPFKCTHQDLC 6 4 72CSWAARCTHQDLC 7 1 73 CSHPYRCTHQDLC 8 2 74 CSHPFRCTHQDLC 9 1 75CSWPYRCTHQDLC 10 3 76 CRFPFKCTHQDLC 11 1 77 CSFPFRCTHQDLC 12 2 78CSLPFRCTHQDLC 13 3 79 CSWPFRCTHQDLC 14 7 80 CSFPIRCTHQDLC 15 1 81CSLPFKCTHQDLC 16 1 82 CSLPFRCTHQDLC 17 4 83 CSYPIRCTHQDLC 18 2 84CSWSARCTHQDLC 19 10 85 CSLPFKCTHQDLC 20 1 86 CSYPFRCTHQDLC 21 1 87CSFPYKCTHQDLC 22 1 88 CSFPWRCTHQDLC 23 1 89 CSWHARCTHQDLC 24 1 90CSLPFRCTHQDLC 25 2 91 CSYPYRCTHQDLC 26 2 92 CSWWARCTHQDLC 27 1 93CSWPYKCTHQDLC 28 1 94 CSFLYKCTHQDLC 29 2 95 CSLPIRCTHQDLC 30 1 96CSMPYRCTHQDLC 31 2 97 CSIPFKCTHQDLC 32 1 98 CSYPWRCTHQDLC 33 1 99CSFPFWCTHQDLC 34 1 100 CSFSYKCTHQDLC 35 1 101 CSWSYRCTHQDLC 36 1 102CSFMYKCTHQDLC 37 1 103 CSQVVGCTHQDLC 38 1 104 CSWPYHCTHQDLC 39 1 105CSLFDHCTHQDLC 40 1 106 CSHRRWCTHQDLC 41 1 107 CSWQARCTHQDLC 42 1 108

TABLE 18 Abundance of particular motif in each output. Output sequenceswere analysed according to the stringency of selection. The % of aparticular motif in the output from a particular stringency selectionwas calculated. Relative abundance of species from different selectionstringencies WPSR WPAR WSAR WPFR WPYR FPYR FPFR Stringency High 34 17 37 3 7 7 31 19 3 3 3 3 19 41 13 3 0 0 3 9 26 22 7 0 4 7 4 23 6 6 6 0 1613 Low 11 4 11 4 0 4 11

TABLE 19 Top 50 Kallikrein binders containing a fixed WPARmotif. The WPAR sequence was fixed in a bicyclelibrary with positions 1, 6, 7, 8, 9 & 10randomised. Kallikrein selection output sequenceswere isolated and assayed for Kallikrein binding.The sequences were ranked according to Kallikreinbinding. The sequence of peptide 06-34-03 wasisolated from selection and is highlighted in red.Trends are visible in the second loop of WPAR-containing Kallikrein-binding peptides. BINDING ASSAY RANK SEQUENCESIGNAL SEQ ID NO. 1 CNWPARCTHQDLC 116 109 2 CSWPARCTHQDLC 110  87 3CHWPARCTHQDLC 110 110 4 CPWPARCTHQDLC 107 111 5 CSWPARCTHADLC 100 112 6CSWPARCTHDDLC  93 113 7 CAWPARCTHTDLC  92 114 8 CQWPARCTHTDLC   91 115 9CLWPARCTHQDLC  90 116 10 CTWPARCTHTDLC  88 117 11 CHWPARCTHQELC  85 11812 CAWPARCTHDDLC  84 119 13 CSWPARCTHTDLC  83 120 14 CAWPARCTHVDLC  82121 15 CAWPARCTHTDFC  80 122 16 CMWPARCMHQDLC  79 123 17 CAWPARCTHADLC 79 124 18 CQWPARCMHQDMC  75 125 19 CQWPARCTHSDLC  74 126 20CLWPARCTHADLC  74 127 21 CRWPARCTHQDLC  73 128 22 CQWPARCMHQELC  73 12923 CTWPARCLHQDLC  73 130 24 CSWPARCTHSHLC  72 131 25 CVWPARCTHQDLC  71132 26 CTWPARCTHADLC  71 133 27 CHWPARCMHQDLC  71 134 28 CPWPARCTHTDLC 70 135 29 CAWPARCTHYDLC  70 136 30 CPWPARCTHQNLC  69 137 31CSWPARCTHTELC  69 138 32 CAWPARCMHDDLC  69 139 33 CSWPARCLHTELC  68 14034 CSWPARCIHQDLC  68 141 35 CTWPARCTHTDMC  67 142 36 CAWPARCTHTHLC  66143 37 CAWPARCLHADMC  66 144 38 CAWPARCLHQDWC  63 145 39 CDWPARCMHQEFC 63 146 40 CAWPARCTHQTMC  61 147 41 CTWPARCLHQHMC  61 148 42CSWPARCVHQDMC  61 149 43 CEWPARCLHTDLC  60 150 44 CLWPARCLTIELC  59 15145 CSWPARCTHAEMC   59S 152 46 CRWPARCTHTDLC  59 153 47 CTWPARCTHQAFC  59154 48 CSWPARCTHSDLC  59 155 49 CSWPARCTHDDLC  59 156 50 CPWPARCLHTDLC 58 157

TABLE 20 The output sequences from Kallikrein selections with fixed WPARin the 1^(st) loop, and their associated Kallikrein binding assaysignals, were grouped according to their derivation from ‘THxxL’ motif(A), or ‘xHxDL’ motif (B). The average binding assay signal for allmembers of a given group was calculated. Groups containing precisely thegiven motif are highlighted green; examples of groups with either onemore or one less change away from the motif are also shown. A B Groupsbased on xHxDL motif Groups based on THxxL motif Motif Binding MotifBinding Motif Binding Motif Binding Motif Binding Motif Binding THxDL78.5 xHxDL 70.9 xHxxL 56.1 THxDL 78.5 THxxL 61.0 MHxxx 56.5 MHxDL 66.7xHxDM 52.8 xHxxM 46.3 MHxDL 66.7 MHxxL 55.0 THxxx 53.4 LHxDL 58.0 xHxEL51.7 xHxxF 43.8 LHxDL 58.0 LHxxL 47.9 LHxxx 44.4 THxEL 55.9 xHxHM 45.4xHxxW 33.2 THxEL 55.9 THxxM 46.3 LTxxx 33.8 THxHL 52.5 xHxNL 46.2 xTxxL31.1 THxHL 52.5 LHxxM 39.0 LHxEL 52.3 xHxDW 44.2 xHxxE 19.3 LHxEL 52.3LTxxL 38.4 THxNL 51.9 xHxHL 44.2 THxNL 51.9 THxDW 44.9 xHxFL 40.4 THxFL40.4 LHxDW 44.7 xTxEL 39.9 LTxEL 39.9 MHxEL 42.4 xTxDL 39.4 LTxDL 39.4LTxEL 39.9 xHxQL 39.0 LHxSL 37.4 LTxDL 39.4 xHxAL 35.5 THxAL 35.5 LHxQL39.0 xHxSL 35.0 LHxHL 34.0 LHxDM 38.0 xTxSL 33.1 LTxSL 33.1 LHxSL 37.4xHxYL 29.2 THxSL 28.9 THxAL 35.5 LHxHL 34.0 LTxSL 33.1 THxSL 28.9

TABLE 21 Affinities and stabilities of WPAR motif variants. ObservableKi (nM) (human in rat plasma, Peptide kallikrein) for days Ac-(06-34-18)WPAR 0.17 2 Ac-(06-34-18) FPAR 6.28 2 Ac-(06-34-18) WPYR 0.41 2Ac-(06-34-18) WPSR 0.44 2 Ac-(06-34-18) FPYR 0.46 2

TABLE 22 Effect of substitutions on Phe2/Tyr4 with hydrophobic analoguesKi (nM) (human Peptide kallikrein) Ac-(06-34-18) Phe2 Tyr4 0.46Ac-(06-34-18) Phe2 Cha4 0.91 Ac-(06-34-18) Phe2 3Pal4 2.57 Ac-(06-34-18)Phe2 4Pal4 2.20 Ac-(06-34-18) Phe2 1Nal4 13.5 Ac-(06-34-18) Phe2 2Nal47.27 Ac-(06-34-18) Phe2 4,4-BPal4 10.5 Ac-(06-34-18) 3Pal2 Tyr4 0.91Ac-(06-34-18) 4Pal2 Tyr4 3.56 Ac-(06-34-18) Cha2 Tyr4 1.87

TABLE 23 Summary of the effect of Arg5 substitutions and Aze3 onAc-06-34-18(TMB)-NH2 Phe2Tyr4. Observable t_(1/2) IC50 Ki (nM) in rat(hrs) in (nM) (human plasma, rat (rat Peptide kallikrein) for days¹plasma² kallikrein)³ Ac-(06-34-18) 0.46 2 0.9 13.8 Phe2 Tyr4Ac-(06-34-18) Phe2 0.77 >10 12.2 19.7 Tyr4 HomoArg5 Ac-(06-34-18) Phe20.40 >10 5.0 2.5 Tyr4 4GuanPhe5 Ac-(06-34-18) Phe2 3.56 >10 >20 60.2Tyr4 NMeArg5 Ac-(06-34-18) Phe2 0.12 nd nd nd Aze3 Tyr4 HomoArg5Ac-(06-34-18) Phe2 0.36 nd nd nd Aze3Tyr4 4GuanPhe5 Ac-(06-34-18) Phe20.97 nd nd nd Aze3Tyr4 NMeArg5 ¹Comparative stabilities estimatedaccording to method #1. ²The true half-life of peptide stabilities inrat plasma was determined according to method #3. ³IC50 values arerelative, not absolute.

TABLE 24a Effect of single amino acid substitutions at position 6. Themodifications were tested both in the context of Ac-(06-34-18) Phe2 Tyr4(left column) and Ac-(06-34-18) (right column). Residue 6 Ki (nM) Ki(nM) Modifications Ac-(06-34-18) Phe2 Tyr4 Ac-(06-34-18) Phe6 14.90 ndIle6 1.46 nd Phg6 0.30 0.18 tBuGly6 3.76 0.49 Chg6 0.50 0.13

TABLE 24b Effect of single amino acid substitutions at position 7. Themodifications were tested only in the context of Ac-(06-34-18) Phe2Tyr4. Residue 7 Modifications Ki (nM) F (Ki_(var)/Ki_(WT)) Ac-(06-34-18)Phe2 Tyr4 (wild type) 0.42 1 Ac-(06-34-18) Phe2 Tyr4 Phe7 43 102Ac-(06-34-18) Phe2 Tyr4 Thi7 34 80 Ac-(06-34-18) Phe2 Tyr4 4ThiAz7 14 33Ac-(06-34-18) Phe2 Tyr4 1,2,4-TriAz7 455 1084 Ac-(06-34-18) Phe2 Tyr44FuAla7 17 39 Ac-(06-34-18) Phe2 Tyr4 2Pal7 53 126 Ac-(06-34-18) Phe2Tyr4 3Pal7 130 308 Ac-(06-34-18) Phe2 Tyr4 4Pal7 79 187 Ac-(06-34-18)Phe2 Tyr4 Dap7 246 586 Ac-(06-34-18) Phe2 Tyr4 Agp7 672 1599Ac-(06-34-18) Phe2 Tyr4 Agb7 2253 5365 Ac-(06-34-18) Phe2 Tyr4 His1Me710 23 Ac-(06-34-18) Phe2 Tyr4 His3Me7 292 696

TABLE 24c Effect of single amino acid substitutions at position 8. Themodifications were tested only in the context of Ac-(06-34-18) Phe2Tyr4. Residue 8 Modifications Ki (nM) F (Ki_(var)/Ki_(WT)) Ac-(06-34-18)Phe2 Tyr4 Thr8 4.23 10 Ac-(06-34-18) Phe2 Tyr4 Thr7 Thr8 Glu9 81.02 193Ac-(06-34-18) Phe2 Tyr4 Asp8 3.83 9 Ac-(06-34-18) Phe2 Tyr4 Glu8 2.53 6Ac-(06-34-18) Phe2 Tyr4 Dap8 5.49 13 Ac-(06-34-18) Phe2 Tyr4 bHGln816.83 40

TABLE 25a α-N-methyl amino acid scan in Loop2. Resultant potencies arecompared to WT, and peptide fragments observed after incubation in humanplasma are noted. Fragments Ki F (human N-methyl scan (nM)(Ki_(var)/Ki_(WT)) plasma) Ac-(06-34-18) Phe2 Tyr4 NMe-Leu6 57.41 137-R, -FPYR Ac-(06-34-18) Phe2 Tyr4 NMe-His7 63.53 151 -R, -FPYRAc-(06-34-18) Phe2 Tyr4 NMe-Gln8 62.77 149 -R, -FPYR Ac-(06-34-18) Phe2Tyr4 NMe-Asp9 44.29 105 -R, -FPYR Ac-(06-34-18) Phe2 Tyr4 NMe- 586.381396 -R, -FPYR Leu10

TABLE 25b Peptide backbone modifications in Loop2. Peptide BackboneModifications Ki (nM) F (Ki_(var)/Ki_(WT)) Ac-(06-34-18) Phe2 Tyr4N-His7 218 519 Ac-(06-34-18) Phe2 Tyr4 Ala(ψCH2NH)6 1.47 4

TABLE 26a Comparative potencies of loop2 plasma protease stabilityenhanced candidates towards human and rat plasma kallikrein. Ki (nM) Ki(nM) (human (rat Suitable candidates kallikrein) kallikrein)Ac-(06-34-18) Phe2 Tyr4 (wild type) 0.90 nd Ac-(06-34-18) Phe2 Tyr4D-Asp9 3.89 32.02 Ac-(06-34-18) D-Ala9 0.87 nd Ac-(06-34-18) Phe2 Tyr4Phg6 0.30 7.23 Ac-(06-34-18) Phe2 Tyr4 tBuGly6 3.76 218.50 Ac-(06-34-18)Phe2 Tyr4 Chg6 0.50 9.09 Ac-(06-34-18) Phe2 Tyr4 Glu8 2.53 ndAc-(06-34-18) Phe2 Tyr4 Ala(ψCH2NH)6 1.47 43.56

TABLE 26b Comparative potencies of loop 1 and loop 2 - modified 06-34-18derivatives. Note the inclusion of Aze3 (affinity enhancing) and HArg5(plasma stability enhancing) modifications in loop 1, and the stabilityenhancing modifications in loop 2. Ki (nM) Ki (nM) (human (rat Loop1 andLoop 2 mulitvariants kallikrein) kallikrein) Ac-(06-34-18) Phe2 Aze3Tyr4 HArg5 D-Asp9 0.90 14 Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Glu8 1.6523 Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg56 0.44 41 Ala(ψCH2NH) Ac-(06-34-18)Phe2 Aze3 Tyr4 tBuGly6 1.91 193 Ac-(06-34-18) Aze3 HArg5 Phg6 0.52 57Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 Phg6 0.32 12

TABLE 27 Pharmacokinetic parameters of (Ac-(06-34-18) Phe2 Aze3 Tyr4HArg5 Ala(ψCH2NH)6, Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 D-Asp9, andAc-(06-34-18) All D control in rat. Due to divergent peptideconcentrations of Ac-(06-34-18) Phe2 Aze3 Tyr4 HArg5 D-Asp9 in twoindividual rats, clearance rates and volumes of distribution could notbe calculated with confidence. Clearance based Volume of t_(1/2) onAUC_(0-∞) distribution based elimination Bicyclic Peptide (ml/min/kg) onAUC_(0-∞) (L/kg) (min) (Ac-(06-34-18) Phe2 6.2 0.44 48 Aze3 Tyr4 HArg5Ala(ψCH₂NH)6 Ac-(06-34-18) Phe2 nd nd 27 Aze3 Tyr4 HArg5 D- Asp9)Ac-(06-34-18) All D, 4.5 0.3  54 control

The invention claimed is:
 1. A peptide ligand specific for humanKallikrein comprising a polypeptide comprising at least three reactivegroups, separated by two loop sequences, and a molecular scaffold whichforms covalent bonds with the reactive groups of the polypeptide suchthat two polypeptide loops are formed on the molecular scaffold, whereinthe loops of the peptide ligand comprise five amino acids and wherein afirst loop comprises the motif G_(r)xWx₁Ax₂G_(r) (SEQ ID NO: 3) orG_(r)xFx₁Yx₂G_(r) (SEQ ID NO: 6) and a second loop comprises the motifG_(r)A(ψCH₂NH)H^(Q)/_(TX)LG_(r) (SEQ ID NO: 212), wherein G_(r) at theC-terminal of the motif in the first loop is the G_(r) at the N-terminalof the motif in the second loop, and wherein x is any amino acid, x₁ isproline or azetidine carboxylic acid, x₂ is arginine, N-methyl arginine,homoarginine, or guanidylphenylalanine, and Gr represents cysteine.
 2. Apeptide ligand according to claim 1, wherein in the first loop x₁ isazetidine carboxylic acid; and/or in the first loop x₂ is N-methylarginine or homoarginine or guanidylphenylalanine.
 3. A peptide ligandaccording to claim 2, wherein the polypeptide comprises a first loopwhich comprises the motif G_(r)xFx₁Yx₂G_(r) (SEQ ID NO: 6), wherein x₁is azetidine carboxylic acid and/or x₂ is N-methyl arginine orhomoarginine or guanidylphenylalanine.
 4. The peptide according to claim1, which comprises: Cys Ser1 Phe 2 Aze3 Tyr4 HArg5 Cys Ala(ψCH₂NH)6 His7Gln8 Asp9 Leu10 Cys (SEQ ID NO: 201).
 5. The peptide ligand according toclaim 1, which comprises:


6. The peptide ligand according to claim 1, which is attached to anantibody.